Antibodies, variable domains &amp; chains tailored for human use

ABSTRACT

The invention relates to the provision of antibody therapeutics and prophylactics that are tailored specifically for human use. The present invention provides libraries, vertebrates and cells, such as transgenic mice or rats or transgenic mouse or rat cells. Furthermore, the invention relates to methods of using the vertebrates to isolate antibodies or nucleotide sequences encoding antibodies. Antibodies, heavy chains, polypeptides, nucleotide sequences, pharmaceutical compositions and uses are also provided by the invention.

CROSS REFERENCE

This is a Continuation Application of PCT/GB2012/052296 filed Sep. 18, 2012, which claims priority to GB 1116122.1 filed Sep. 19, 2011, GB 1116120.5 filed Sep. 19, 2011, GB 1203257.9 filed Feb. 24, 2012, GB 1204592.8 filed Mar. 15, 2012, GB 1205702.2 filed Mar. 29, 2012, GB 1208749.0 filed May 18, 2012 and GB 1211692.7 filed Jul. 2, 2012, all of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the provision of antibody therapeutics and prophylactics that are tailored specifically for human use.

The present invention provides libraries, vertebrates and cells, such as transgenic mice or rats or transgenic mouse or rat cells. Furthermore, the invention relates to methods of using the vertebrates to isolate antibodies or nucleotide sequences encoding antibodies. Antibodies, heavy chains, polypeptides, nucleotide sequences, pharmaceutical compositions and uses are also provided by the invention.

BACKGROUND

The state of the art provides non-human vertebrates (e.g., mice and rats) and cells comprising transgenic immunoglobulin loci, such loci comprising human variable (V), diversity (D) and/or joining (J) segments, and optionally human constant regions. Alternatively, endogenous constant regions of the host vertebrate (e.g., mouse or rat constant regions) are provided in the transgenic loci. Methods of constructing such transgenic vertebrates and use of these to generate antibodies and nucleic acids thereof following antigen immunisation are known in the art, e.g., see U.S. Pat. No. 7,501,552 (Medarex), U.S. Pat. No. 5,939,598 (Abgenix), U.S. Pat. No. 6,130,364 (Abgenix), WO02/066630 (Regeneron), WO2011004192 (Genome Research Limited), WO2009076464, WO2009143472 and WO2010039900 (Ablexis), the disclosures of which are explicitly incorporated herein. Such transgenic loci in the art include varying amounts of the human V(D) J repertoire. Existing transgenic immunoglobulin loci are based on a single human DNA source. The potential diversity of human antibody variable regions in non-human vertebrates bearing such transgenic loci is thus confined.

The inventors considered that it would be desirable to tailor the genomes of these transgenic non-human vertebrates (and thus antibody and antibody chain products of these) to address the variability—and commonality—in the natural antibody gene usage of humans. The inventors wanted to do this in order to better address human use of antibody-based therapeutic and prophylactic drugs.

It would be desirable also to provide for novel and potentially expanded repertoire and diversity of human variable regions in transgenic immunoglobulin loci and non-human vertebrates harbouring these, as well as in antibodies produced following immunisation of such animals.

SUMMARY OF THE INVENTION

The present invention has been developed from extensive bioinformatics analysis of natural antibody gene segment distributions across a myriad of different human populations and across more than two thousand samples from human individuals. The inventors have undertaken this huge task to more thoroughly understand and design non-human vertebrate systems and resultant antibodies to better address human medical therapeutics as a whole, as well as to enable rational design to address specific ethnic populations of humans. Using such rational design, the inventors have constructed transgenic non-human vertebrates and isolated antibodies, antibody chains and cells expressing these in a way that yields products that utilise gene segments that have been purposely included on the basis of the human bioinformatics analysis. The examples illustrate worked experiments where the inventors isolated many cells and antibodies to this effect.

The invention also relates to synthetically-extended & ethnically-diverse superhuman immunoglobulin gene repertoires. The present invention thus provides for novel and potentially expanded synthetic immunoglobulin diversities, thus providing a pool of diversity from which human antibody therapeutic leads can be selected. This expanded pool is useful when seeking to find antibodies with desirable characteristics, such as relatively high affinity to target antigen without the need for further affinity maturation (e.g., using laborious in vitro techniques such as phage or ribosome display), or improved biophysical characteristics, or to address targets and new epitopes that have previously been difficult to address with antibodies are not reached by prior antibody binding sites.

The invention also provides for diversity that is potentially biased towards variable gene usage common to members of a specific human population, which is useful for generating antibodies for treating and/or preventing diseases or conditions within such population. This ability to bias the antibody repertoire allows one to tailor antibody therapeutics with the aim of more effectively treating and/or preventing disease or medical conditions in specific human populations.

The present inventors realised the possibility of providing immunoglobulin gene segments from disparate sources in transgenic loci, in order to provide for novel and potentially-expanded antibody diversities from which antibody therapeutics (and antibody tool reagents) could be generated. This—opens up the potential of transgenic human-mouse/rat technologies to the possibility of interrogating different and possibly larger antibody sequence-spaces than has hitherto been possible.

In rationally designing transgenic antibody loci, as well as antibodies and antibody chains, the inventors also realised that a relatively long HCDR3 length (at least 20 amino acids) is often desirable to address epitopes. For example, naturally-occurring antibodies have been isolated from humans infected with infectious disease pathogens, such antibodies having a long HCDR3 length. Neutralizing antibodies have been found in this respect. A long HCDR3 length would be desirable to address other antigens (e.g., receptor clefts or enzyme active sites), not just limited to infectious disease pathogens, and thus the inventors realised the general desirability of the possibility of engineering transgenic loci to be able to produce long HCDR3 antibodies and heavy chains. The inventors, through laborious execution of bioinformatics on in excess of 2000 human DNA samples via the 1000 Genomes project together with rational sequence choices, identified that the inclusion of the specific human gene segment variant JH6*02 is desirable for producing long HCDR3 antibodies and chains.

Additional rational design and bioinformatics has led the inventors to realise that specific human constant region variants are conserved across many diverse human populations. The inventors realised that this opens up the possibility of making a choice to humanize antibodies, chains and variable domains by using such specific constant regions in products, rather than arbitrarily choosing the human constant region (or a synthetic version of a human constant region). This aspect of the invention also enables one to tailor antibody-based drugs to specific human ethnic populations, thereby more closely matching drug to patient (and thus disease setting) than has hitherto been performed. It can be a problem in the state of the art that antibodies are humanized with an arbitrary choice of human constant region (presumably derived from one (often unknown) ethnic population or non-naturally occurring) that does not function as well in patients of a different human ethnic population. This is important, since the constant region has the major role in providing antibody effector functions, e.g., for antibody recycling, cellular and complement recruitment and for cell killing.

To this end, in a first configuration of the invention, there is provided

First Configuration

A non-human vertebrate or vertebrate cell (optionally an ES cell or antibody-producing cell) comprising a genome having a superhuman immunoglobulin heavy chain human VH and/or D and/or J gene repertoire.

A non-human vertebrate or vertebrate cell (optionally an ES cell or antibody-producing cell) comprising a genome having a superhuman immunoglobulin light chain human VL gene repertoire; optionally wherein the vertebrate or cell is according to the first configuration.

A non-human vertebrate or vertebrate cell (optionally an ES cell or antibody-producing cell) whose genome comprises a transgenic immunoglobulin locus (e.g., a heavy chain locus or a light chain locus), said locus comprising immunoglobulin gene segments according to the first and second human immunoglobulin gene segments (optionally V segments) as mentioned below operably connected upstream of an immunoglobulin constant region; optionally wherein the genome is homozygous for said transgenic immunoglobulin locus;

optionally wherein the immunoglobulin locus comprises more than the natural human complement of functional V gene segments; and/or optionally wherein the immunoglobulin locus comprises more than the natural human complement of functional D gene segments; and/or optionally wherein the immunoglobulin locus comprises more than the natural human complement of functional J gene segments.

A transgenic non-human vertebrate (e.g., a mouse or rat) or vertebrate cell (optionally an ES cell or antibody-producing cell) whose genome comprises a transgenic immunoglobulin locus comprising a plurality of human immunoglobulin gene segments operably connected upstream of a non-human vertebrate constant region for the production of a repertoire of chimaeric antibodies, or chimaeric light or heavy chains, having a non-human vertebrate constant region and a human variable region; wherein the transgenic locus comprises one or more human immunoglobulin V gene segments, one or more human J gene segments and optionally one or more human D gene segments, a first (optionally a V segment) of said gene segments and a second (optionally a V segment) of said gene segments being different and derived from the genomes of first and second human individuals respectively, wherein the individuals are different; and optionally not related; optionally wherein the immunoglobulin locus comprises more than the natural human complement of functional V gene segments; and/or

optionally wherein the immunoglobulin locus comprises more than the natural human complement of functional D gene segments; and/or optionally wherein the immunoglobulin locus comprises more than the natural human complement of functional J gene segments.

A transgenic non-human vertebrate (e.g., a mouse or rat) or vertebrate cell (optionally an ES cell or antibody-producing cell) whose genome comprises first and second transgenic immunoglobulin loci, each locus comprising a plurality of human immunoglobulin gene segments operably connected upstream of a non-human vertebrate constant region for the production of a repertoire of chimaeric antibodies, or chimaeric light or heavy chains, having a non-human vertebrate constant region and a human variable region;

wherein (i) the first transgenic locus comprises one or more human immunoglobulin V gene segments, one or more human J gene segments and optionally one or more human D gene segments, (ii) the second transgenic locus comprises one or more human immunoglobulin V gene segments, one or more human J gene segments and optionally one or more human D gene segments; and (iii) wherein a first (optionally a V) gene segment of said first locus and a second (optionally a V) gene segment of said second gene locus are different and derived from the genomes of first and second human individuals respectively, wherein the individuals are different; and optionally not related; optionally wherein the first and second loci are on different chromosomes (optionally chromosomes with the same chromosome number) in said genome; optionally wherein each immunoglobulin locus comprises more than the natural human complement of functional V gene segments; and/or optionally wherein each immunoglobulin locus comprises more than the natural human complement of functional D gene segments; and/or optionally wherein each immunoglobulin locus comprises more than the natural human complement of functional J gene segments.

A method of constructing a cell (e.g., an ES cell) according to the invention, the method comprising

-   (a) identifying functional V and J (and optionally D) gene segments     of the genome sequence of a (or said) first human individual; -   (b) identifying one or more functional V and/or D and/or J gene     segments of the genome sequence of a (or said) second human     individual, wherein these additional gene segments are not found in     the genome sequence of the first individual; -   (c) and constructing a transgenic immunoglobulin locus in the cell,     wherein the gene segments of (a) and (b) are provided in the locus     operably connected upstream of a constant region.

In one embodiment, the gene segment(s) in step (b) are identified from an immunoglobulin gene database selected from the 1000 Genomes, Ensembl, Genbank and IMGT databases.

Throughout this text, Genbank is a reference to Genbank release number 185.0 or 191.0; the 1000 Genomes database is Phase 1, release v3, 16 Mar. 2012; the Ensembl database is assembly GRCh37.p8 (Oct. 4, 2012); the IMGT database is available at www.imgt.org.

In one embodiment, the first and second human individuals are members of first and second ethnic populations respectively, wherein the populations are different, optionally wherein the human immunoglobulin gene segment derived from the genome sequence of the second individual is low-frequency (optionally rare) within the second ethnic population.

This configuration of the invention also provides a method of making a transgenic non-human vertebrate (e.g., a mouse or rat), the method comprising

-   (a) constructing an ES cell (e.g., a mouse C57BL/6N, C57BL/6J, 129S5     or 129Sv strain ES cell) by carrying out the method above; -   (b) injecting the ES cell into a donor non-human vertebrate     blastocyst (e.g., a mouse C57BL/6N, C57BU6J, 129S5 or 129Sv strain     blastocyst); -   (c) implanting the blastocyst into a foster non-human vertebrate     mother (e.g., a C57BL/6N, C57BL/6J, 129S5 or 129Sv strain mouse);     and -   (d) obtaining a child from said mother, wherein the child genome     comprises a transgenic immunoglobulin locus.

In one embodiment, the invention provides a method of isolating an antibody that binds a predetermined antigen (e.g., a bacterial or viral pathogen antigen), the method comprising immunizing a non-human vertebrate according to the invention.

Second Configuration

A library of antibody-producing transgenic cells whose genomes collectively encode a repertoire of antibodies, wherein

-   (a) a first transgenic cell expresses a first antibody having a     chain encoded by a first immunoglobulin gene, the gene comprising a     first variable domain nucleotide sequence produced following     recombination of a first human unrearranged immunoglobulin gene     segment; -   (b) a second transgenic cell expresses a second antibody having a     chain encoded by a second immunoglobulin gene, the second gene     comprising a second variable domain nucleotide sequence produced     following recombination of a second human unrearranged     immunoglobulin gene segment, the first and second antibodies being     non-identical; -   (c) the first and second gene segments are different and derived     from the genome sequences of first and second human individuals     respectively, wherein the individuals are different; and optionally     not related; -   (d) wherein the cells are non-human vertebrate (e.g., mouse or rat)     cells.

In one embodiment, the first and second human individuals are members of first and second ethnic populations respectively, wherein the populations are different; optionally wherein the ethnic populations are selected from those identified in the 1000 Genomes database.

In another embodiment, the second human immunoglobulin gene segment is a polymorphic variant of the first human immunoglobulin gene segment; optionally wherein the second gene segment is selected from the group consisting of a gene segment in any of Tables 1 to 7 and 9 to 14 below (e.g., selected from Table 13 or Table 14), e.g., the second gene segment is a polymorphic variant of VH1-69.

Third Configuration An isolated antibody having

-   (a) a heavy chain encoded by a nucleotide sequence produced     following recombination in a transgenic non-human vertebrate cell of     an unrearranged human immunoglobulin V gene segment with a human D     and human J segment, optionally with affinity maturation in said     cell, wherein one of the gene segments is derived from the genome of     an individual from a first human ethnic population; and the other     two gene segments are derived from the genome of an individual from     a second, different, human ethnic population, and wherein the     antibody comprises heavy chain constant regions of said non-human     vertebrate (e.g., rodent, mouse or rat heavy chain constant     regions); and/or -   (b) a light chain encoded by a nucleotide sequence produced     following recombination in a transgenic non-human vertebrate cell of     an unrearranged human immunoglobulin V gene segment with a human J     segment, optionally with affinity maturation in said cell, wherein     one of the gene segments is derived from the genome of an individual     from a first human ethnic population (optionally the same as the     first population in (a)); and the other gene segment is derived from     the genome of an individual from a second, different, human ethnic     population (optionally the same as the second population in (a)),     and wherein the antibody comprises light chain constant regions of     said non-human vertebrate (e.g., rodent, mouse or rat heavy light     constant regions); -   (c) Optionally wherein each variable domain of the antibody is a     human variable domain. -   (d) Optionally wherein the heavy chain constant regions are     gamma-type constant regions.

The invention also provides an isolated nucleotide sequence encoding the antibody, optionally wherein the sequence is provided in an antibody expression vector, optionally in a host cell.

The invention also provides a method of producing a human antibody, the method comprising replacing the non-human vertebrate constant regions of the antibody of the third configuration with human antibody constant regions.

The invention also provides a pharmaceutical composition comprising an antibody according to the third configuration, or an antibody produced according to the method above and a diluent, excipient or carrier; optionally wherein the composition is provided in a container connected to an IV needle or syringe or in an IV bag.

The invention also provides an antibody-producing cell that expresses the second antibody recited in any one of the configurations.

In an alternative configuration, the invention contemplates the combination of nucleotide sequences of first and second immunoglobulin gene segments (e.g., two or more polymorphic variants of a particular human germline VH or VL gene segment) to provide a synthetic gene segment. Such synthetic gene segment is used, in one embodiment, to build a transgenic immunoglobulin locus, wherein the synthetic gene segment is provided in combination with one or more human variable and J regions (and optionally one or more human D regions) operably connected upstream of a constant region. When provided in the genome of a non-human vertebrate or cell (e.g., mouse or rat cell, e.g., ES cell), the invention provides for superhuman gene segment diversity. The sequences to be combined can be selected from gene segments that have been observed to be commonly used in human antibodies raised against a particular antigen (e.g., a flu antigen, such as haemaglutinin). By combining the sequences, the synthetic gene segment may recombine in vivo to produce an antibody that is well suited to the treatment and/or prevention of a disease or condition (e.g., influenza) mediated by said antigen.

Fourth Configuration

A non-human vertebrate (optionally a mouse or a rat) or vertebrate cell whose genome comprises an immunoglobulin heavy chain locus comprising human gene segment JH6*02, one or more VH gene segments and one or more D gene segments upstream of a constant region; wherein the gene segments in the heavy chain locus are operably linked to the constant region thereof so that the mouse is capable of producing an antibody heavy chain produced by recombination of the human JH6*02 with a D segment and a VH segment.

A non-human vertebrate cell (optionally a mouse cell or a rat cell) whose genome comprises an immunoglobulin heavy chain locus comprising human gene segment JH6*02, one or more VH gene segments and one or more D gene segments upstream of a constant region; wherein the gene segments in the heavy chain locus are operably linked to the constant region thereof for producing (e.g., in a subsequent progeny cell) an antibody heavy chain produced by recombination of the human JH6*02 with a D segment and a VH segment.

A heavy chain (e.g., comprised by an antibody) isolated from a vertebrate of the invention wherein the heavy chain comprises a HCDR3 of at least 20 amino acids.

A method for producing a heavy chain, VH domain or an antibody specific to a target antigen, the method comprising immunizing a non-human vertebrate according to the invention with the antigen and isolating the heavy chain, VH domain or an antibody specific to a target antigen or a cell producing the heavy chain, VH domain or an antibody, wherein the heavy chain, VH domain or an antibody comprises a HCDR3 that is derived from the recombination of human JH6*02 with a VH gene segment and a D gene segment.

A heavy chain, VH domain or an antibody produced by the method.

A B-cell or hybridoma expressing a heavy chain VH domain that is identical to the VH domain of the heavy chain.

A nucleic acid encoding the VH domain of the heavy chain of claim 22, 23 or 28, or encoding the heavy chain.

A vector (e.g., a CHO cell or HEK293 cell vector) comprising the nucleic acid; optionally wherein the vector is in a host cell (e.g., a CHO cell or HEK293 cell).

A pharmaceutical composition comprising the antibody, heavy chain or VH domain (e.g., comprised by an antibody), together with a pharmaceutically-acceptable excipient, diluent or a medicament (e.g., a further antigen-specific variable domain, heavy chain or antibody).

The antibody, heavy chain or VH domain (e.g., comprised by an antibody) as above for use in medicine.

The use of an antibody, heavy chain or VH domain (e.g., comprised by an antibody) as above in the manufacture of a medicament for treating and/or preventing a medical condition in a human.

Fifth Configuration

A method of producing an antibody heavy chain, the method comprising

-   (a) providing an antigen-specific heavy chain variable domain; and -   (b) combining the variable domain with a human heavy chain constant     region to produce an antibody heavy chain comprising (in N- to     C-terminal direction) the variable domain and the constant region;     wherein     the human heavy chain constant region is an IGHG1ref, IGHG2ref,     IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref or IGHG4a constant     region.

An antibody comprising a human heavy chain, the heavy chain comprising a variable domain that is specific for an antigen and a constant region that is an IGHG1 ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref or IGHG4a constant region. Optionally, the variable domain comprises mouse-pattern AID somatic mutations.

A polypeptide comprising (in N- to C-terminal direction) a leader sequence, a human variable domain that is specific for an antigen and a human constant region that is an IGHG1 ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref or IGHG4a constant region wherein (i) the leader sequence is not the native human variable domain leader sequence; and/or (ii) the variable domain comprises mouse AID-pattern somatic mutations and/or mouse Terminal deoxynucleotidyl transferase (TdT)-pattern junctional mutations.

A nucleotide sequence encoding (in 5′ to 3′ direction) a leader sequence and a human antibody heavy chain, the heavy chain comprising a variable domain that is specific for an antigen and a constant region that is an IGHG1ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref or IGHG4a constant region; and the leader sequence being operable for expression of the heavy chain and wherein the leader sequence is not the native human variable domain leader sequence.

A nucleotide sequence encoding (in 5′ to 3′ direction) a promoter and a human antibody heavy chain, the heavy chain comprising a variable domain that is specific for an antigen and a constant region that is an IGHG1ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref or IGHG4a constant region; and the promoter being operable for expression of the heavy chain and wherein the promoter is not the native human promoter.

A vector (e.g., a CHO cell or HEK293 cell vector) comprising a IGHG1ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref or IGHG4a constant region nucleotide sequence that is 3′ of a cloning site for the insertion of a human antibody heavy chain variable domain nucleotide sequence, such that upon insertion of such a variable domain sequence the vector comprises (in 5′ to 3′ direction) a promoter, a leader sequence, the variable domain sequence and the constant region sequence so that the vector is capable of expressing a human antibody heavy chain when present in a host cell.

Sixth Configuration

A non-human vertebrate (e.g., a mouse or rat) or a non-human vertebrate cell (e.g., an ES cell or a B-cell) having a genome comprising at least 3 human variable region gene segments of the same type (e.g., at least 3 human VH6-1 gene segments, at least 3 human JH6 gene segments, at least 3 human VK1-39 gene segments, at least 3 human D2-2 gene segments or at least 3 human JK1 gene segments), wherein at least two of the human gene segments are variants that are not identical to each other.

A non-human vertebrate (e.g., a mouse or rat) or a non-human vertebrate cell (e.g., an ES cell or a B-cell) having a genome comprising at least 2 different non-endogenous variable region gene segments of the same type (e.g., at least 2 human VH6-1 gene segments, at least 3 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human D2-2 gene segments or at least 2 human JK1 gene segments) cis at the same Ig locus.

A non-human vertebrate (e.g., a mouse or rat) or a non-human vertebrate cell (e.g., an ES cell or a B-cell) having a genome comprising at least 2 different human variable region gene segments of the same type (e.g., at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human D2-2 gene segments or at least 2 human JK1 gene segments) trans at the same Ig locus; and optionally a third human gene segment of the same type, wherein the third gene segment is cis with one of said 2 different gene segments.

A population of non-human vertebrates (e.g., mice or rats) comprising a repertoire of human variable region gene segments, wherein the plurality comprises at least 2 human variable region gene segments of the same type (e.g., at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human D2-2 gene segments or at least 2 human JK1 gene segments), a first of said different gene segments is provided in the genome of a

first vertebrate of the population, and a second of said different gene segments being provided in the genome of a second vertebrate of the population, wherein the genome of the first vertebrate does not comprise the second gene segment.

A non-human vertebrate (e.g., a mouse or rat) or a non-human vertebrate cell (e.g., an ES cell or a B-cell) having a genome comprising at least 2 different non-endogenous variable region gene segments of the same type (e.g., at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human D2-2 gene segments or at least 2 human JK1 gene segments), wherein the gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations.

A method of enhancing the human immunoglobulin gene diversity of a non-human vertebrate (e.g., a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 3 human variable region gene segments of the same type (e.g., at least 3 human VH6-1 gene segments, at least 3 human JH6 gene segments, at least 3 human VK1-39 gene segments, at least 3 human D2-2 gene segments or at least 3 human JK1 gene segments), wherein at least two of the human gene segments are variants that are not identical to each other.

A method of enhancing the immunoglobulin gene diversity of a non-human vertebrate (e.g., a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 different non-endogenous variable region gene segments of the same type (e.g., at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human D2-2 gene segments or at least 2 human JK1 gene segments) cis at the same Ig locus.

A method of enhancing the immunoglobulin gene diversity of a non-human vertebrate (e.g., a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 different human variable region gene segments of the same type (e.g., at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human D2-2 gene segments or at least 2 human JK1 gene segments) trans at the same Ig locus; and optionally a third human gene segment of the same type, wherein the third gene segment is cis with one of said 2 different gene segments.

A method of providing an enhanced human immunoglobulin variable region gene segment repertoire, the method comprising providing a population of non-human vertebrates (e.g., a mouse or rat) comprising a repertoire of human variable region gene segments, wherein the method comprises providing at least 2 different human variable region gene segments of the same type (e.g., at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human D2-2 gene segments or at least 2 human JK1 gene segments), wherein a first of said different gene segments is provided in the genome of a first vertebrate of the population, and a second of said different gene segments is provided in the genome of a second vertebrate of the population, wherein the genome of the first vertebrate does not comprise the second gene segment.

A method of enhancing the human immunoglobulin gene diversity of a non-human vertebrate (e.g., a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 different non-endogenous variable region gene segments of the same type (e.g., at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human D2-2 gene segments or at least 2 human JK1 gene segments), wherein the gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations.

A method of enhancing the human immunoglobulin gene diversity of a non-human vertebrate (e.g., a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 human variable region gene segments of the same type (e.g., at least 2 human VH6-1 gene segments, at least 2 human JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2 human D2-2 gene segments or at least 2 human JK1 gene segments), wherein the gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations; optionally wherein at least 2 or 3 of said different gene segments are provided at the same Ig locus in said genome.

A non-human vertebrate (e.g., a mouse or rat) or a non-human vertebrate cell (e.g., an ES cell or a B-cell) having a genome comprising first and second human Ig locus gene segments of the same type (e.g., first and second human JH6 gene segments; or first and second IgG2 gene segments; or first and second human JA7 gene segments), wherein the first gene segment is a gene segment selected from any one of Tables 1 and 9 to 14 (e.g., selected from Table 13 or Table 14) (e.g., IGHJ6-a) and the second gene segment is the corresponding reference sequence.

A population of non-human vertebrates (e.g., mice or rats) comprising first and second human Ig locus gene segments of the same type (e.g., first and second human JH6 gene segments; or first and second IgG2 gene segments; or first and second human JA7 gene segments), wherein the first gene segment is a gene segment selected from any one of Tables 1 and 9 to 14 (e.g., selected from Table 13 or Table 14) (e.g., IGHJ6-a) and the second gene segment is the corresponding reference sequence, wherein the first gene segment is provided in the genome of a first vertebrate of the population, and the second gene segment is provided in the genome of a second vertebrate of the population.

A method of enhancing the human immunoglobulin gene diversity of a non-human vertebrate (e.g., a mouse or rat), the method comprising providing the vertebrate with a genome comprising first and second human Ig locus gene segments of the same type (e.g., first and second human JH6 gene segments; or first and second IgG2 gene segments; or first and second human JA7 gene segments), wherein the first gene segment is a gene segment selected from any one of Tables 1 and 9 to 14 (e.g., selected from Table 13 or Table 14) (e.g., IGHJ6-a) and the second gene segment is the corresponding reference sequence.

In one aspect of this configuration, the invention relates to human D gene segment variants as described further below.

In one aspect of this configuration, the invention relates to human V gene segment variants as described further below.

In one aspect of this configuration, the invention relates to human J gene segment variants as described further below.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 to 3: Schematic illustrating a protocol for producing recombineered BAC vectors to add V gene segments into a mouse genome;

FIG. 4: Schematic illustrating a protocol for adding V gene segments to a mouse genome using sequential recombinase mediated cassette exchange (sRMCE); and

FIG. 5 (in 4 parts): Alignment of 13 IGHV1-69 variants showing the variable (V) coding region only. Nucleotides that differ from VH1-69 variant *01 are indicated at the appropriate position whereas identical nucleotides are marked with a dash. Where nucleotide changes result in amino acid differences, the encoded amino acid is shown above the corresponding triplet. Boxed regions correspond to CDR1, CDR2 and CDR3 as indicated.

FIG. 6 is a schematic illustrating gene segment diversity and the effect of including variant variants in cis according to the invention:—

-   (a) Situation in a normal person: Recombination on the same     chromosome limits combinations of variants, for instance the     antibody gene V4-4 can only be recombined within variant 1 to form     for instance for instance V4-4-D-J6 or V4-4-D-J2A. Similarly the     variant V4-4A can't be recombined with either J6 or J2A from variant     1 and can only be joined with J-genes from variant 2 to form     V4-4A-D-J6A and V4-4A-D-J2. V4-4-J2/J6 complexity=4. -   (b) Situation in a transgenic mouse: Only one variant is provided so     the genome is limited. V4-4-J6/J2 complexity=2. -   (c) Supra mouse of the invention: The variants are added in cis and     thus can be recombined in every combination, expanding the     repertoire. For instance V4-4 can be combined with J6A, J6, J2A or     J2 and similarly V4-4A can be recombined with these same J-genes.     The V4-4-J6/J2 complexity=8, which in this simple example is double     that of a person and 4× that of a mouse with a single variant.

FIG. 7: Alignment of human JH6*02 variants. Nucleotides that differ from JH6*01 are indicated at the appropriate position whereas identical nucleotides are marked with a dash. Where nucleotide changes result in amino acid differences, the encoded amino acid is shown above. Accession numbers (e.g., J00256) are shown to the left of the IMGT variant name.

FIG. 8: Alignment of JH sequences from various species.

FIG. 9: Codon Table

FIG. 10: BAC database extract

BRIEF DESCRIPTION OF THE TABLES

-   Table 1: Human IgH V Polymorphic Variants -   Table 2: Human IgH D Polymorphic Variants -   Table 3: Human IgH J Polymorphic Variants -   Table 4: Human Ig Vk Polymorphic Variants -   Table 5: Human Ig VA Polymorphic Variants -   Table 6: Human IgH Jk Polymorphic Variants -   Table 7: Human IgH JA Polymorphic Variants -   Table 8: 1000 Genomes Project Human Populations -   Table 9: Immunoglobulin Gene Usage in Human Antibody Responses to     Infectious Disease Pathogens -   Table 10A: Human IgH JH5 Variant Occurrences -   Table 10B: Non-Synonymous Human IgH JH5 Variants -   Table 11A: Human IgH JH6 Variant Occurrences -   Table 11B: Non-Synonymous Human IgH JH6 Variants -   Table 12A: Human IgH JH2 Variant Occurrences -   Table 12B: Non-Synonymous Human IgH JH2 Variants -   Table 13: Variant Frequency Analyses & Human Population     Distributions -   Table 14: Frequent Human Variant Distributions -   Table 15: Human Gene Segment Usage: Heavy Chain Repertoires From     Naive Non-Human Vertebrates -   Table 16: Human Gene Segment Usage: Heavy Chain Repertoires From     Immunised Non-Human Vertebrates -   Table 17: Human Gene Segment Usage: Heavy Chain Repertoires From     Antigen-Specific Hybridomas -   Table 18: Sequence Correlation Table -   Table 19: Summary Of Function Correlated With Human Gamma Constant     Region Sub-Type -   Table 20: Gene Segments Prevalent In Few Human Populations -   Table 21: Genomic and sequence information

DETAILED DESCRIPTION OF THE INVENTION

A suitable source of JH6*02 and other human DNA sequences for use in the invention will be readily apparent to the skilled person. For example, it is possible to collect a DNA sample from a consenting human donor (e.g., a cheek swab sample as per the Example herein) from which can be obtained suitable DNA sequences for use in constructing a locus of the invention. Other sources of human DNA are commercially available, as will be known to the skilled person. Alternatively, the skilled person is able to construct gene segment sequence by referring to one or more databases of human Ig gene segment sequences disclosed herein.

An example source for human V, D and J gene segments according to the invention are Bacterial Artificial Chromosomes (RPCI-11 BACs) obtained from Roswell Park Cancer Institute (RPCI)/Invitrogen. See http://bacpac.chori.org/hmale11.htm which describes the BACs as follows: —

“RPCI-11 Human Male BAC Library

The RPCI-11 Human Male BAC Library (Osoegawa et al., 2001) was constructed using improved cloning techniques (Osoegawa et al., 1998) developed by Kazutoyo Osoegawa. The library was generated by Kazutoyo Osoegawa. Construction was funded by a grant from the National Human Genome Research Institute (NHGRI, NIH) (#1R01RG01165-03). This library was generated according to the new NHGRI/DOE “Guidance on Human Subjects in Large-Scale DNA Sequencing . . . .

“Male blood was obtained via a double-blind selection protocol. Male blood DNA was isolated from one randomly chosen donor (out of 10 male donors)”.

-   -   Osoegawa K, Mammoser A G, Wu C, Frengen E, Zeng C, Catanese J J,         de Jong P J; Genome Res. 2001 March; 11(3):483-96; “A bacterial         artificial chromosome library for sequencing the complete human         genome”;     -   Osoegawa, K., Woon, P. Y., Zhao, B., Frengen, E., Tateno, M.,         Catanese, J. J, and de Jong, P. J. (1998); “An Improved Approach         for Construction of Bacterial Artificial Chromosome Libraries”;         Genomics 52, 1-8.

Superhuman Immunoglobulin Gene Repertoires

The invention relates to synthetically-extended & ethnically-diverse superhuman immunoglobulin gene repertoires. The human immunoglobulin repertoires are beyond those found in nature (i.e., “Superhuman”), for example, they are more diverse than a natural human repertoire or they comprise combinations of human immunoglobulin gene segments from disparate sources in a way that is non-natural. Thus, the repertoires of the invention are “superhuman” immunoglobulin repertoires, and the invention relates to the application of these in transgenic cells and non-human vertebrates for utility in producing chimaeric antibodies (with the possibility of converting these into fully-human, isolated antibodies using recombinant DNA technology). The present invention thus provides for novel and potentially expanded synthetic immunoglobulin diversities, which provides for a pool of diversity from which antibody therapeutic leads (antibody therapeutics and antibody tool reagents) can be selected. This opens up the potential of transgenic human-mouse/rat technologies to the possibility of interrogating different and possibly larger antibody sequence-spaces than has hitherto been possible. To this end, in one embodiment, the invention provides a SUPERHUMAN MOUSE™ (aka SUPRA-MOUSE™) and a SUPERHUMAN RAT™ (aka SUPRA-RAT™)

In developing this thinking, the present inventors have realised the possibility of mining the huge genetics resources now available to the skilled person thanks to efforts such as the HapMap Project, 1000 Genomes Project and sundry other immunoglobulin gene databases (see below for more details). Thus, in some embodiments, the inventors realised the application of these genome sequencing developments in the present invention to generate synthetically-produced and ethnically-diverse artificial immunoglobulin gene repertoires. In one aspect, the inventors realised that such repertoires are useful for the production of antibodies having improved affinity and/or biophysical characteristics, and/or wherein the range of epitope specificities produced by means of such repertoire is novel, provides for antibodies to epitopes that have hitherto been intractable by prior transgenic immunoglobulin loci or difficult to address.

The present invention provides libraries, vertebrates and cells, such as transgenic mice or rats or transgenic mouse or rat cells. Furthermore, the invention relates to methods of using the vertebrates to isolate antibodies or nucleotide sequences encoding antibodies. Antibodies, nucleotide sequences, pharmaceutical compositions and uses are also provided by the invention.

Variation Analysis

The present inventors have realized methods and antibody loci designs that harness the power of genetic variation analysis. The reference human genome provides a foundation for experimental work and genetic analysis of human samples. The reference human is a compilation of the genomes from a small number of individuals and for any one segment of the genome a high quality single reference genome for one of the two chromosomes is available. Because the reference genome was assembled from a series of very large insert clones, the identity of these clones is known. Accordingly, experimental work with human genomic DNA is usually conducted on the clones from which the reference sequence was derived.

Individual humans differ in their sequence and recently several individuals have had their genomes sequenced, for instance James Watson and Craig Venter. Comparison of the genome sequence of these individuals has revealed differences between their sequences and the reference genome in both coding and non-coding parts of the genome, approximately 1 in 1000 bases are different. Some variants will be significant and contribute to differences between individuals. In extreme cases these will result in genetic disease. Variation can be implicated in differing responses to drugs administered to human patients, e.g., yielding an undesirable lowering of patient response to treatment.

The 1000-Genomes Project has the objective of identifying the most frequent variations in the human genome. This public domain project involved sequencing the genomes of more than 1000 individuals from diverse ethnic groups, comparing these sequences to the reference and assembling a catalogue of variants. This has enabled the annotation of variants in coding regions, but because this sequence wasn't derived from large clones of DNA, the analysis of the sequence from diploid individuals can't discriminate the distribution of the variation between the maternal and paternally inherited chromosomes. Where more than one variant is identified in a protein coding gene, it is not possible to illuminate the distribution of the pattern of variants in each version of the protein. For example, if two variants are detected in different positions of the same protein in an individual, this could have resulted from one copy with two variants and none in the other or each copy could have just one variant. To illuminate the sequence of real proteins, the 1000-Genome Project has sequenced mother-father-child trios. This allows one to “phase” the sequence variants, in other words identify blocks of sequence that are inherited from one or other parent and deconvolute the variants.

To further understand the variation within the 1000-genome set a tool has been developed that can identify the significant variants (defined as non-synonymous amino acid changes) from a region of DNA from the phased data in the 1000-genome data set. This tool has been made available online http://www.1000genomes.org/variation-pattern-finder. This tool allows an investigator to download non-synonymous variation delimited between specific coordinates. The downloaded files are configured as individual genotypes, but the data is phased so the haplotype information and the frequencies of specific halotypes in different populations can be extracted.

The inventors' analysis of the 1000-genome data for the individual human coding segments of the C, V D and J genes from the heavy and light chains reveals that there is significant variation in these segments. Individuals will usually have two different heavy chain alleles and also different light chain alleles at both kappa and lambda loci. The repertoire of antibodies that can be generated from each allele will be different. This variation will contribute to a better or differing immune response to certain antigens.

Humanized mice that have hitherto been generated with immunoglobulin heavy and light chain loci contain just one type of immunoglobulin locus. Even if these mice contain a full human heavy chain locus, the variation will be less than contained in a typical human because only one set of C, V, D and J genes are available, while a typical human would have two sets.

The inventors have devised ways to improve on this limitation when constructing transgenic non-human vertebrates and cells for human antibody and variable region production in vivo.

Mice can be generated with two different loci, each engineered to have a different repertoire of V, D and J segments. This could be in a single mouse or two or more separate mouse strains and would be analogous to or beyond the repertoire found in a normal human. The engineering of such a mouse would go beyond the repertoire described in humanized mice to date which only have one set of alleles.

However, the inventors also realized that this also has limitations, because the different loci would not normally interact to shuffle V, D and J variants between loci. This same limitation is also inherent in a human, thus this system does not utilize the advantage of recombining variants in all combinations.

To go beyond the normal repertoire in humans and take advantage of combinations of C, V, D and J variants the inventors decided, in one embodiment, to provide these on the same chromosome in cis. See FIG. 6. These loci would be characterized by having more than the normal number of J, D or V genes. For example n=6 for the J genes, but including one J6 variant and one J2 variant would increase this to n=8. This could be combined with additional variants for the D and V genes, for example. By detailed analysis of the 1000-Genomes database, the inventors have devised a collection of candidate polymorphic human variant gene segments, e.g., JH gene segments (e.g., see the examples), that can be built into the design of transgenic heavy and light chain loci in mice for expressing increasingly diverse and new, synthetic repertoires of human variable regions. Moreover, by utilizing naturally-occurring human variant gene segments, as per embodiments of the invention, this addresses compatibility with human patients since the inventors analysis has drawn out candidate variants that are naturally conserved and sometimes very prevalent amongst human ethnic populations. Additionally this enables one to tailor the configurations of the invention to provide for antibody-based drugs that better address specific human ethnic populations.

In an example according to any configuration of the invention, loci (and cells and vertebrates comprising these) are provided in which gene segments from different human populations are used. This is desirable to increase antibody gene diversity to better address more diverse human patients. In an example, the gene segments are from first and second different human populations respectively, and thus the second gene segment is found in the second human population, but not so (or rarely) in the first human population. Rarely means, for example, that the gene segment is found in 5, 4, 3, 2, or 1 or zero individuals in the first population in the 1000 Genomes database. For example, the first gene segment may be shown as present in a first population by reference to Table 13 or 14 herein, the second gene segment may be shown as present in the second population by reference to Table 13 and not in the first population. Optionally, the first gene segment may also be shown as being present in the second population by reference to Table 13 or 14.

In any configuration or aspect of the invention, where a V gene segment is used, this may be used optionally with the native leader sequence. For example, use of genomic DNA (e.g., from BACs as in the examples) will mean that the native leader will be used for each V gene segment incorporated into the locus and genomes of the invention. In an alternative, the skilled person may wish to inert a non-native leader sequence together with one or more of the V gene segments. Similarly, in any configuration or aspect of the invention, where a V gene segment is used, this may be used optionally with the native 5′ UTR sequence. For example, use of genomic DNA (e.g., from BACs as in the examples) will mean that the native 5′ UTR sequence will be used for each V gene segment incorporated into the locus and genomes of the invention. In an alternative, the skilled person may wish to exclude the native 5′ UTR sequence.

The Present Invention Provides, in a First Configuration (a) Superhuman Heavy Chain Gene Repertoires

-   -   A non-human vertebrate or vertebrate cell (optionally an ES cell         or antibody-producing cell) comprising a genome having a         superhuman immunoglobulin heavy chain human VH and/or D and/or J         gene repertoire.     -   In one aspect the cell of the invention is an embryonic stem         cell. For example, the ES cell is derived from the mouse         C57BL/6N, C57BL/6J, 129S5 or 129Sv strain. In one aspect the         non-human vertebrate is a rodent, suitably a mouse, and cells of         the invention, are rodent cells or ES cells, suitably mouse ES         cells. The ES cells of the present invention can be used to         generate animals using techniques well known in the art, which         comprise injection of the ES cell into a blastocyst followed by         implantation of chimaeric blastocystys into females to produce         offspring which can be bred and selected for homozygous         recombinants having the required insertion. In one aspect the         invention relates to a transgenic animal comprised of ES         cell-derived tissue and host embryo derived tissue. In one         aspect the invention relates to genetically-altered subsequent         generation animals, which include animals having a homozygous         recombinants for the VDJ and/or VJ regions.     -   The natural human immunoglobulin gene segment repertoire         consists of (see e.g., www.imgt.org):—     -   VH: total-125; functional-41 DH: total-27; functional-23 JH:         total-8; functional-6     -   Vk: total-77; functional-38 Jk: total-5; functional-5     -   V lambda: total-75; functional-31     -   J lambda: total-7; functional-5     -   In one embodiment, the vertebrate or cell genome comprises a         transgenic immunoglobulin heavy chain locus comprising a         plurality of human immunoglobulin VH gene segments, one or more         human D gene segments and one or more human J gene segments,         wherein the plurality of VH gene segments consists of more than         the natural human repertoire of functional VH gene segments;         optionally wherein the genome is homozygous for said transgenic         heavy chain locus.     -   In one embodiment of the vertebrate or cell, the VH gene         repertoire consists of a plurality of VH gene segments derived         from the genome sequence of a first human individual,         supplemented with one or more different VH gene segments derived         from the genome sequence of a second, different human         individual. Optionally the D and J segments are derived from the         genome sequence of the first human individual. Optionally the VH         gene segments from the genome sequence of the second individual         are selected from the VH gene segments listed in Table 1, 13         or 14. In this way, the locus provides a superhuman repertoire         of D gene segments.     -   Optionally the individuals are not related. Individuals are “not         related” in the context of any configuration or aspect of the         invention, for example, if one of the individuals does not         appear in a family tree of the other individual in the same         generation or going back one, two, three or four generations.         Alternatively, are not related, for example, if they do not         share a common ancestor in the present generation or going back         one, two, three or four generations.     -   In one embodiment of the vertebrate or cell, the transgenic         locus comprises more than 41 functional human VH gene segment         species, and thus more than the natural human functional         repertoire. Optionally the locus comprises at least 42, 43, 44,         45, 46, 47, 48, 49 or 50 functional human VH gene segment         species (e.g., wherein the locus comprises the full functional         VH repertoire of said first individual supplemented with one or         more VH gene segments derived from the genome sequence of the         second human individual and optionally with one or more VH gene         segments derived from the genome sequence of a third human         individual). In this way, the locus provides a superhuman         repertoire of VH gene segments that is useful for generating a         novel gene and antibody diversity for use in therapeutic and         tool antibody selection.     -   In one embodiment of the vertebrate or cell, the transgenic         locus comprises a first VH gene segment derived from the genome         sequence of the first individual and a second VH gene segment         derived from the genome sequence of the second individual,         wherein the second VH gene segment is a polymorphic variant of         the first VH gene segment. For example, the VH gene segments are         polymorphic variants of VH1-69 as illustrated in the examples         below. Optionally the locus comprises a further polymorphic         variant of the first VH gene segment (e.g., a variant derived         from the genome sequence of a third human individual). In this         way, the locus provides a superhuman repertoire of VH gene         segments.     -   In one embodiment of the vertebrate or cell, the genome         (alternatively or additionally to the superhuman VH diversity)         comprises a transgenic immunoglobulin heavy chain locus         comprising a plurality of human immunoglobulin VH gene segments,         a plurality of human D gene segments and one or more human J         gene segments, wherein the plurality of D gene segments consists         of more than the natural human repertoire of functional D gene         segments. Optionally the genome is homozygous for said         transgenic heavy chain locus.     -   In one embodiment of the vertebrate or cell, the D gene         repertoire consists of a plurality of D gene segments derived         from the genome sequence of a (or said) first human individual,         supplemented with one or more different D gene segments derived         from the genome sequence of a (or said) second, different human         individual. Optionally the individuals are not related.         Optionally the J segments are derived from the genome sequence         of the first human individual. Optionally the D gene segments         from the genome sequence of the second individual are selected         from the D gene segments listed in Table 2, 13 or 14. In this         way, the locus provides a superhuman repertoire of D gene         segments.     -   In one embodiment of the vertebrate or cell, the transgenic         locus comprises more than 23 functional human D gene segment         species; optionally wherein the locus comprises at least 24, 25,         26, 27, 28, 29, 30 or 31 functional human D gene segment species         (e.g., wherein the locus comprises the full functional D         repertoire of said first individual supplemented with one or         more D gene segments derived from the genome sequence of the         second human individual and optionally with one or more D gene         segments derived from the genome sequence of a third human         individual). In this way, the locus provides a superhuman         repertoire of D gene segments.     -   In one embodiment of the vertebrate or cell, the transgenic         locus comprises a first D gene segment derived from the genome         sequence of the first individual and a second D gene segment         derived from the genome sequence of the second individual,         wherein the second D gene segment is a polymorphic variant of         the first D gene segment. Optionally the locus comprises a         further polymorphic variant of the first D gene segment (e.g., a         variant derived from the genome sequence of a third human         individual). In this way, the locus provides a superhuman         repertoire of D gene segments.     -   In one embodiment of the vertebrate or cell (alternatively or         additionally to the superhuman VH and/or JH diversity), the         genome comprises a (or said) transgenic immunoglobulin heavy         chain locus comprising a plurality of human immunoglobulin VH         gene segments, one or more human D gene segments and a plurality         of human JH gene segments, wherein the plurality of J gene         segments consists of more than the natural human repertoire of         functional J gene segments; optionally wherein the genome is         homozygous for said transgenic heavy chain locus.     -   In one embodiment of the vertebrate or cell, the JH gene         repertoire consists of a plurality of J gene segments derived         from the genome sequence of a (or said) first human individual,         supplemented with one or more different J gene segments derived         from the genome sequence of a (or said) second, different human         individual. Optionally the individuals are not related.         Optionally D segments are derived from the genome sequence of         the first human individual. Optionally the J gene segments from         the genome sequence of the second individual are selected from         the J gene segments listed in Table 3 13 or 14. In this way, the         locus provides a superhuman repertoire of JH gene segments.     -   In one embodiment of the vertebrate or cell, the transgenic         locus comprises more than 6 functional human JH gene segment         segments. Optionally the locus comprises at least 7, 8, 9, 10,         11, 12, 13, 14, 15, or 16 functional human JH gene segments         (e.g., wherein the locus comprises the full functional JH         repertoire of said first individual supplemented with one or         more JH gene segments derived from the genome sequence of the         second human individual and optionally with one or more JH gene         segments derived from the genome sequence of a third human         individual). In this way, the locus provides a superhuman         repertoire of JH gene segments.     -   In one embodiment of the vertebrate or cell, the transgenic         locus comprises a first JH gene segment derived from the genome         sequence of the first individual and a second JH gene segment         derived from the genome sequence of the second individual,         wherein the second JH gene segment is a polymorphic variant of         the first JH gene segment. Optionally the locus comprises a         further polymorphic variant of the first JH gene segment (e.g.,         a variant derived from the genome sequence of a third human         individual). In this way, the locus provides a superhuman         repertoire of JH gene segments.

-   (b) Superhuman Light Chain Gene Repertoires     -   The first configuration of the invention also provides:—     -   A non-human vertebrate or vertebrate cell (optionally an ES cell         or antibody-producing cell) comprising a genome having a         superhuman immunoglobulin light chain human VL gene repertoire.         Optionally the vertebrate or cell comprises a heavy chain         transgene according to aspect (a) of the first configuration.         Thus, superhuman diversity is provided in both the heavy and         light chain immunoglobulin gene segments in the cell and         vertebrate. For example, the genome of the cell or vertebrate is         homozygous for the heavy and light chain transgenes and         endogenous antibody expression is inactivated. Such a vertebrate         is useful for immunisation with a predetermined antigen to         produce one or more selected antibodies that bind the antigen         and have human variable regions resulting from recombination         within the superhuman gene segment repertoire. This provides         potentially for a novel antibody and gene sequence space from         which to select therapeutic, prophylactic and tool antibodies.     -   In one embodiment of aspect (b) of the first configuration, the         vertebrate or cell genome comprises     -   (i) a transgenic immunoglobulin kappa light chain locus         comprising a plurality of human immunoglobulin VK gene segments         and one or more human J gene segments, wherein the plurality of         VK gene segments consists of more than the natural human         repertoire of functional VK gene segments; optionally wherein         the genome is homozygous for said transgenic kappa light chain         locus; and/or     -   (ii) a transgenic immunoglobulin lambda light chain locus         comprising a plurality of human immunoglobulin VA gene segments         and one or more human J gene segments, wherein the plurality of         VA gene segments consists of more than the natural human         repertoire of functional VA gene segments; optionally wherein         the genome is homozygous for said transgenic lambda light chain         locus.         -   In this way, the locus provides a superhuman repertoire of             VL gene segments. In one embodiment of the vertebrate or             cell,     -   (i) the VK gene repertoire consists of a plurality of VK gene         segments derived from the genome sequence of a first human         individual, supplemented with one or more VK gene segments         derived from the genome sequence of a second, different human         individual; optionally wherein the individuals are not related;         optionally wherein the J segments are derived from the genome         sequence of the first human individual; and optionally wherein         the VK gene segments from the genome sequence of the second         individual are selected from the VK gene segments listed in         Table 4, 13 or 14; and     -   (i) the VA gene repertoire consists of a plurality of VA gene         segments derived from the genome sequence of a first human         individual, supplemented with one or more VA gene segments         derived from the genome sequence of a second, different human         individual; optionally wherein the individuals are not related;         optionally wherein the J segments are derived from the genome         sequence of the first human individual; and optionally wherein         the VA gene segments from the genome sequence of the second         individual are selected from the VA gene segments listed in         Table 5, 13 or 14.

In this way, the locus provides a superhuman repertoire of VL gene segments.

In one embodiment of the vertebrate or cell,

-   -   the kappa light transgenic locus comprises more than 38         functional human VK gene segment species; optionally wherein the         locus comprises at least 39, 40, 41, 42, 43, 44, 45, 46, 47 or         48 functional human VK gene segment species (e.g., wherein the         locus comprises the full functional VK repertoire of said first         individual supplemented with one or more VK gene segments         derived from the genome sequence of the second human individual         and optionally with one or more VK gene segments derived from         the genome sequence of a third human individual); and     -   the lambda light transgenic locus comprises more than 31         functional human VA gene segment species; optionally wherein the         locus comprises at least 32, 33, 34, 35, 36, 37, 38, 39, 40 or         41 functional human VA gene segment species (e.g., wherein the         locus comprises the full functional VA repertoire of said first         individual supplemented with one or more VA gene segments         derived from the genome sequence of the second human individual         and optionally with one or more VA gene segments derived from         the genome sequence of a third human individual).

In this way, the locus provides a superhuman repertoire of VL gene segments.

In one embodiment of the vertebrate or cell,

-   -   the kappa light transgenic locus comprises a first VK gene         segment derived from the genome sequence of the first individual         and a second VK gene segment derived from the genome sequence of         the second individual, wherein the second VK gene segment is a         polymorphic variant of the first VK gene segment; optionally         wherein the locus comprises a further polymorphic variant of the         first VK gene segment (e.g., a variant derived from the genome         sequence of a third human individual); and     -   the lambda light transgenic locus comprises a first VA gene         segment derived from the genome sequence of the first individual         and a second VA gene segment derived from the genome sequence of         the second individual, wherein the second VA gene segment is a         polymorphic variant of the first VA gene segment; optionally         wherein the locus comprises a further polymorphic variant of the         first VA gene segment (e.g., a variant derived from the genome         sequence of a third human individual).

In this way, the locus provides a superhuman repertoire of VL gene segments.

In one embodiment of the vertebrate or cell, the genome comprises a (or said) transgenic immunoglobulin light chain locus comprising a plurality of human immunoglobulin VL gene segments and a plurality of human JL gene segments, wherein the plurality of J gene segments consists of more than the natural human repertoire of functional J gene segments; optionally wherein the genome is homozygous for said transgenic heavy chain locus.

In one embodiment of the vertebrate or cell,

-   (i) the JK gene repertoire consists of a plurality of JK gene     segments derived from the genome sequence of a (or said) first human     individual, supplemented with one or more JK gene segments derived     from the genome sequence of a (or said) second, different human     individual; optionally wherein the individuals are not related;     optionally wherein the VK segments are derived from the genome     sequence of the first human individual; optionally wherein the JK     gene segments from the genome sequence of the second individual are     selected from the JK gene segments listed in Table 6, 13 or 14; and -   (ii) the JK gene repertoire consists of a plurality of JAgene     segments derived from the genome sequence of a (or said) first human     individual, supplemented with one or more JA gene segments derived     from the genome sequence of a (or said) second, different human     individual; optionally wherein the individuals are not related;     optionally wherein the VA segments are derived from the genome     sequence of the first human individual; optionally wherein the JA     gene segments from the genome sequence of the second individual are     selected from the JA gene segments listed in Table 7, 13 or 14.     -   In this way, the locus provides a superhuman repertoire of JL         gene segments. In one embodiment of the vertebrate or cell, -   (i) the transgenic light chain locus comprises more than 5     functional human JK gene segment species; optionally wherein the     locus comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15     functional human JK gene segment species (e.g., wherein the locus     comprises the full functional JK repertoire of said first individual     supplemented with one or more JK gene segments derived from the     genome sequence of the second human individual and optionally with     one or more JK gene segments derived from the genome sequence of a     third human individual); and/or     -   (i) the transgenic light chain locus comprises more than 5         functional human JA gene segment species; optionally wherein the         locus comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15         functional human JA gene segment species (e.g., wherein the         locus comprises the full functional JA repertoire of said first         individual supplemented with one or more JA gene segments         derived from the genome sequence of the second human individual         and optionally with one or more JA gene segments derived from         the genome sequence of a third human individual).     -   In this way, the locus provides a superhuman repertoire of JL         gene segments.     -   In one embodiment of the vertebrate or cell,     -   (i) the kappa light transgenic locus comprises a first JK gene         segment derived from the genome sequence of the first individual         and a second JK gene segment derived from the genome sequence of         the second individual, wherein the second JK gene segment is a         polymorphic variant of the first JK gene segment; optionally         wherein the locus comprises a further polymorphic variant of the         first JK gene segment (e.g., a variant derived from the genome         sequence of a third human individual); and     -   (ii) the lambda light transgenic locus comprises a first JA gene         segment derived from the genome sequence of the first individual         and a second JA gene segment derived from the genome sequence of         the second individual, wherein the second JK gene segment is a         polymorphic variant of the first JA gene segment; optionally         wherein the locus comprises a further polymorphic variant of the         first JA gene segment (e.g., a variant derived from the genome         sequence of a third human individual).     -   In this way, the locus provides a superhuman repertoire of JL         gene segments. Further aspects of the first configuration are         described below.

The Present Invention Provides, in a Second Configuration

A library of antibody-producing transgenic cells whose genomes collectively encode a repertoire of antibodies, wherein

-   (a) a first transgenic cell expresses a first antibody having a     chain (e.g., heavy chain) encoded by a first immunoglobulin gene,     the gene comprising a first variable domain nucleotide sequence     produced following recombination of a first human unrearranged     immunoglobulin gene segment (e.g., a VH); -   (b) a second transgenic cell expresses a second antibody having a     chain (e.g., a heavy chain) encoded by a second immunoglobulin gene,     the second gene comprising a second variable domain nucleotide     sequence produced following recombination of a second human     unrearranged immunoglobulin gene segment (e.g., a VH), the first and     second antibodies being non-identical; -   (c) the first and second gene segments are different and derived     from the genome sequences of first and second human individuals     respectively, wherein the individuals are different; and optionally     not related; -   (d) wherein the cells are non-human vertebrate (e.g., mouse or rat)     cells (e.g., B-cells or hybridomas).

In one embodiment, the library is provided in vitro. In another embodiment, the library is provided in vivo by one or a plurality of transgenic non-human vertebrates. For example, the or each vertebrate is according to any aspect of the first configuration of the invention.

In one embodiment, the library encodes an antibody repertoire of from 10 to 109 antibodies, for example, 10, 20, 30, 40, 50, 100 or 1000 to 108; or 10, 20, 30, 40, 50, 100 or 1000 to 107; or 10, 20, 30, 40, 50, 100 or 1000 to 106; or 10, 20, 30, 40, 50, 100 or 1000 to 105; or 10, 20, 30, 40, 50, 100 or 1000 to 104 antibodies. In an example, library encodes an antibody repertoire of at least 103, 104, 105, 106, 107, 108, 109, or 1010 antibodies.

The first variable domain nucleotide sequence is produced following recombination of the first human unrearranged immunoglobulin gene segment with one or more other immunoglobulin gene segments (for example, human immunoglobulin gene segments). For example, where the first gene segment is a VH, the first variable domain nucleotide sequence (a VH domain) is produced following recombination of the VH with a human D and JH segments in vivo, optionally with somatic hypermutation, in the first transgenic cell or an ancestor thereof. For example, where the first gene segment is a VL, the first variable domain nucleotide sequence (a VL domain) is produced following recombination of the VL with a human JL segment in vivo, optionally with somatic hypermutation, in the first transgenic cell or an ancestor thereof.

The second variable domain nucleotide sequence is produced following recombination of the second human unrearranged immunoglobulin gene segment with one or more other immunoglobulin gene segments (for example, human immunoglobulin gene segments). For example, where the second gene segment is a VH, the second variable domain nucleotide sequence (a VH domain) is produced following recombination of the VH with a human D and JH segments in vivo, optionally with somatic hypermutation, in the second transgenic cell or an ancestor thereof. For example, where the second gene segment is a VL, the second variable domain nucleotide sequence (a VL domain) is produced following recombination of the VL with a human JL segment in vivo, optionally with somatic hypermutation, in the second transgenic cell or an ancestor thereof.

The first and second gene segments are respectively derived from genome sequences of first and second human individuals. In one example, such a gene segment is isolated or cloned from a sample cell taken from said individual using standard molecular biology techniques as know to the skilled person. The sequence of the gene segment may be mutated (e.g., by the introduction of up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotide changes) prior to use in the present invention. In another example, a gene segment is derived by identifying a candidate human immunoglobulin gene segment in a database (see guidance below) and a nucleotide sequence encoding a gene segment for use in the present invention is made by reference (e.g., to be identical or a mutant with up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotide changes to the reference sequence) to the database sequence. The skilled person will be aware of methods of obtaining nucleotide sequences by reference to databases or by obtaining from cellular samples.

In one embodiment of the vertebrate, cell or library of any configuration of the invention, the first and second human individuals are members of first and second ethnic populations respectively, wherein the populations are different. This, therefore, provides for superhuman gene diversity in transgenic loci, cells and vertebrates as per the invention.

Human Populations

Optionally the ethnic populations are selected from those identified in the 1000 Genomes Project of database. In this respect, see Table 8 which provides details of the ethnic populations on which the 1000 Genomes database is based.

N A Rosenberg et al (Science 20 Dec. 2002: vol. 298 no. 5602 2342-2343) studied the genetic structure of human populations of differing geographical ancestry. In total, 52 populations were sampled, these being populations with:

African Ancestry

(Mbuti Pygmies, Biaka Pygmies, San peoples, and speakers of Niger-Kordofanian languages (Bantu, Yoruba or Mandenka populations),

Eurasian Ancestry

(European ancestry (Orcadian, Adygei, Basque, French, Russians, Italians, Sardinian, Tuscan), Middle Eastern ancestry (Mozabite, Bedouin, Druze, Palestinians), Central/South Asian ancestry (Balochl, Brahul, Makrani, Sindhi, Pathan, Burusho, Hazara, Uygur, Kalash)),

East Asian Ancestry

(Han, Dal, Daur, Hezhen, Lahu, Miao, Orogen, She, Tujia, Tu, Xibo, Yi, Mongola, Naxi, Cambodian, Japanese, Yakut), Oceanic ancestry (Melanesian, Papuan); or

Americas Ancestry (Karitiana, Surui, Colombian, Maya, Pima).

The International HapMap Project, Nature, 2003 Dec. 18; 426(6968):789-96, discloses that goal of the HapMap Project: to determine the common patterns of DNA sequence variation in the human genome by determining the genotypes of one million or more sequence variants, their frequencies and the degree of association between them in DNA samples from populations with ancestry from parts of Africa, Asia and Europe. The relevant human populations of differing geographical ancestry include Yoruba, Japanese, Chinese, Northern European and Western European populations. More specifically:—

Utah population with Northern or Western European ancestry (samples collected in 1980 by the Centre d'Etude du Polymorphisme Humain (CEPH)); population with ancestry of Yoruba people from Ibadan, Nigeria; population with Japanese ancestry; and population with ancestry of Han Chinese from China.

The authors, citing earlier publications, suggest that ancestral geography is a reasonable basis for sampling human populations.

A suitable sample of human populations from which the populations used in the present invention are selected is as follows:—

-   (a) European ancestry -   (b) Northern European ancestry; Western European ancestry; Toscani     ancestry; British ancestry, Finnish ancestry or Iberian ancestry. -   (c) More specifically, population of Utah residents with Northern     and/or Western European ancestry; Toscani population in Italia;     British population in England and/or Scotland; Finnish population in     Finland; or Iberian population in Spain. -   (a) East Asian ancestry -   (b) Japanese ancestry; Chinese ancestry or Vietnamese ancestry. -   (c) More specifically, Japanese population in Tokyo, Japan; Han     Chinese population in Beijing, China; Chinese Dai population in     Xishuangbanna; Kinh population in Ho Chi Minh City, Vietnam; or     Chinese population in Denver, Colo., USA. -   (a) West African ancestry -   (b) Yoruba ancestry; Luhya ancestry; Gambian ancestry; or Malawian     ancestry. -   (c) More specifically, Yoruba population in Ibadan, Nigeria; Luhya     population in Webuye, Kenya; Gambian population in Western Division,     The Gambia; or Malawian population in Blantyre, Malawi. -   (a) Population of The Americas -   (b) Native American ancestry; Afro-Caribbean ancestry; Mexican     ancestry; Puerto Rican ancestry; Columbian ancestry; or Peruvian     ancestry. -   (c) More specifically, population of African Ancestry in Southwest     US; population of African American in Jackson, Miss.; population of     African Caribbean in Barbados; population of Mexican Ancestry in Los     Angeles, Calif.; population of Puerto Rican in Puerto Rico;     population of Colombian in Medellin, Colombia; or population of     Peruvian in Lima, Peru. -   (a) South Asian ancestry -   (b) Ahom ancestry; Kayadtha ancestry; Reddy ancestry; Maratha; or     Punjabi ancestry. -   (c) More specifically, Ahom population in the State of Assam, India;     Kayadtha population in Calcutta, India; Reddy population in     Hyderabad, India; Maratha population in Bombay, India; or Punjabi     population in Lahore, Pakistan.

In any configuration of the invention, in one embodiment, each human population is selected from a population marked “(a)” above.

In any configuration of the invention, in another embodiment, each human population is selected from a population marked “(b)” above.

In any configuration of the invention, in another embodiment, each human population is selected from a population marked “(c)” above.

In one embodiment of the library of the vertebrate, cell or library of the invention, the first and second ethnic populations are selected from the group consisting of an ethnic population with

European ancestry, an ethnic population with East Asian, an ethnic population with West African ancestry, an ethnic population with Americas ancestry and an ethnic population with South Asian ancestry.

In one embodiment of the library of the vertebrate, cell or library of the invention, the first and second ethnic populations are selected from the group consisting of an ethnic population with Northern European ancestry; or an ethnic population with Western European ancestry; or an ethnic population with Toscani ancestry; or an ethnic population with British ancestry; or an ethnic population with Icelandic ancestry; or an ethnic population with Finnish ancestry; or an ethnic population with Iberian ancestry; or an ethnic population with Japanese ancestry; or an ethnic population with Chinese ancestry; or an ethnic population Vietnamese ancestry; or an ethnic population with Yoruba ancestry; or an ethnic population with Luhya ancestry; or an ethnic population with Gambian ancestry; or an ethnic population with Malawian ancestry; or an ethnic population with Native American ancestry; or an ethnic population with Afro-Caribbean ancestry; or an ethnic population with Mexican ancestry; or an ethnic population with Puerto Rican ancestry; or an ethnic population with Columbian ancestry; or an ethnic population with Peruvian ancestry; or an ethnic population with Ahom ancestry; or an ethnic population with Kayadtha ancestry; or an ethnic population with Reddy ancestry; or an ethnic population with Maratha; or an ethnic population with Punjabi ancestry.

In one embodiment of any configuration of the vertebrate, cell or library of the invention, the human immunoglobulin gene segment derived from the genome sequence of the second individual is low-frequency (optionally rare) within the second ethnic population. Optionally human immunoglobulin gene segment has a Minor Allele Frequency (MAF) (cumulative frequency) of between 0.5%-5%, optionally less than 0.5%, in the second human population, e.g., as in the 1000 Genomes database.

In one embodiment of any configuration of the vertebrate, cell or library of the invention, the first variable region nucleotide sequence is produced by recombination of the first human immunoglobulin gene segment with a first J gene segment and optionally a first D gene segment, wherein the first human immunoglobulin gene segment is a V gene segment and the V, D and J segments are derived from the first human population, optionally from the genome of one individual

of the first human population.

In one embodiment of the library of the vertebrate, cell or library of the invention, the second variable region nucleotide sequence is produced by recombination of the second human immunoglobulin gene segment with a second J gene segment and optionally a second D gene segment, wherein the second human immunoglobulin gene segment is a V gene segment derived from the second population and the D and/or J segments are derived from the first human population, optionally the D and J gene segments being from the genome of one individual of the first human population.

In one embodiment of the library of the vertebrate, cell or library of the invention, all of the D and J segments that have been recombined with the first and second V gene segments are D and J segments derived from the first human population, optionally the D and J gene segments being from the genome of one individual of the first human population.

In one embodiment of the library, the second human immunoglobulin gene segment is a polymorphic variant of the first human immunoglobulin gene segment; optionally wherein the second gene segment is selected from the group consisting of a gene segment in any of Tables 1 to 7 and 9 to 14 (e.g., selected from Table 13 or 14).

In one embodiment of the library, the first and second human immunoglobulin gene segments are both (i) VH gene segments; (ii) D segments; (iii) J segments (optionally both JH segments, both JK segments or both Ĵ segments); (iv) constant regions (optionally both a gamma constant region, optionally both a C gamma-1 constant region); (v) CH1 regions; (vi) CH2 regions; or (vii) CH3 regions.

The library is, for example, a naive and optionally has a library size of from 10 or 102 to 109 cells. For example, from 10, 20, 30, 40, 50, 100 or 1000 to 108; or 10, 20, 30, 40, 50, 100 or 1000 to 107; or 10, 20, 30, 40, 50, 100 or 1000 to 10s; or 10, 20, 30, 40, 50, 100 or 1000 to 105; or 10, 20, 30, 40, 50, 100 or 1000 to 104 cells.

The library has, for example, been selected against a predetermined antigen and optionally has a library size of from 10 or 102 to 109 cells. For example, from 10, 20, 30, 40, 50, 100 or 1000 to 108; or 10, 20, 30, 40, 50, 100 or 1000 to 107; or 10, 20, 30, 40, 50, 100 or 1000 to 10s; or 10, 20, 30, 40, 50, 100 or 1000 to 105; or 10, 20, 30, 40, 50, 100 or 1000 to 104 cells.

In one embodiment of the library of the invention, said first and second cells are progeny of first and second ancestor non-human vertebrate cells respectively, wherein the first ancestor cell comprises a genome comprising said first human immunoglobulin gene segment; and the second ancestor cell comprises a genome comprising said second human immunoglobulin gene segment.

The invention further provides a library of antibody-producing transgenic cells whose genomes collectively encode a repertoire of antibodies, wherein the library comprises the first and second ancestor cells described above.

The invention further provides a library of hybridoma cells produced by fusion of the library of the invention (e.g., a B-cell library) with fusion partner cells and optionally has a library size of from 10 or 102 to 109 cells. For example, from 10, 20, 30, 40, 50, 100 or 1000 to 108; or 10, 20, 30, 40, 50, 100 or 1000 to 107; or 10, 20, 30, 40, 50, 100 or 1000 to 10s; or 10, 20, 30, 40, 50, 100 or 1000 to 105; or 10, 20, 30, 40, 50, 100 or 1000 to 104 cells. Production of hybridomas is well known to the skilled person. Examples of fusion partners are SP2/0-g14 (obtainable from ECACC), P3XS3-Ag8.S53 (obtainable from LGC Standards; CRL-1580), NS1 and NS0 cells. PEG fusion or electrofusion can be carried out, as is conventional.

The Invention Provides, in a Third Configuration:—

An isolated antibody having

-   (a) a heavy chain encoded by a nucleotide sequence produced     following recombination in a transgenic non-human vertebrate cell of     an unrearranged human immunoglobulin V gene segment with a human D     and human J segment, optionally with affinity maturation in said     cell, wherein one of the gene segments (e.g., VH) is derived from     the genome of an individual from a first human ethnic population;     and the other two gene segments (e.g., D and JH) are derived from     the genome of an individual from a second (e.g., a second and third     respectively), different, human ethnic population, and wherein the     antibody comprises heavy chain constant regions (e.g., C gamma) of     said non-human vertebrate (e.g., rodent, mouse or rat heavy chain     constant regions); and/or -   (b) a light chain encoded by a nucleotide sequence produced     following recombination in a transgenic non-human vertebrate cell of     an unrearranged human immunoglobulin V gene segment with a human J     segment, optionally with affinity maturation in said cell, wherein     one of the gene segments (e.g., VL) is derived from the genome of an     individual from a first human ethnic population (optionally the same     as the first population in (a)); and the other gene segment (e.g.,     JL) is derived from the genome of an individual from a second,     different, human ethnic population (optionally the same as the     second population in (a)), and wherein the antibody comprises light     chain constant regions of said non-human vertebrate (e.g., rodent,     mouse or rat heavy light constant regions); -   (c) Optionally wherein each variable domain of the antibody is a     human variable domain. -   (d) Optionally wherein the heavy chain constant regions are mu- or     gamma-type constant regions.

The invention also provides an isolated nucleotide sequence encoding the antibody of the third configuration, optionally wherein the sequence is provided in an antibody expression vector, optionally in a host cell. Suitable vectors are mammalian expression vectors (e.g., CHO cell vectors or HEK293 cell vectors), yeast vectors (e.g., a vector for expression in Picchia pastoris, or a bacterial expression vector, e.g., a vector for E. coli expression.

The invention also provides a method of producing a human antibody, the method comprising replacing the non-human vertebrate constant regions of the antibody of the third configuration with human antibody constant regions (e.g., a C variant disclosed in table 13 or 18). The skilled person will be aware of standard molecular biology techniques to do this. For example, see Harlow, E. & Lane, D. 1998, 5th edition, Antibodies: A Laboratory Manual, Cold Spring Harbor Lab. Press, Plainview, N.Y.; and Pasqualini and Arap, Proceedings of the National Academy of Sciences (2004) 101:257-259 for standard immunisation. Joining of the variable regions of an antibody to a human constant region can be effected by techniques readily available in the art, such as using conventional recombinant DNA and RNA technology as will be apparent to the skilled person. See e.g. Sambrook, J and Russell, D. (2001, 3'd edition) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, N.Y.).

In one embodiment, the method comprises further making a mutant or derivative of the antibody.

The invention also provides a pharmaceutical composition comprising an antibody according to the third configuration, or a human antibody of the invention and a diluent, excipient or carrier; optionally wherein the composition is provided in a container connected to an IV needle or syringe or in an IV bag.

The invention also provides an antibody-producing cell (e.g., a mammalian cell, e.g., CHO or HEK293; a yeast cell, e.g., P. pastoris; a bacterial cell, e.g., E. coli; a B-cell; or a hybridoma) that expresses the second antibody of the third configuration or the isolated antibody of the invention.

The First Configuration of the Invention Also Provides:—

A non-human vertebrate or vertebrate cell (optionally an ES cell or antibody-producing cell) whose genome comprises a transgenic immunoglobulin locus (e.g., a heavy chain locus or a light chain locus), said locus comprising immunoglobulin gene segments according to the first and second human immunoglobulin gene segments (optionally V segments) described above in connection with the third configuration. The gene segments are operably connected upstream of an immunoglobulin constant region; optionally wherein the genome is homozygous for said transgenic immunoglobulin locus.

Optionally the immunoglobulin locus comprises more than the natural human complement of functional V gene segments; and/or

Optionally wherein the immunoglobulin locus comprises more than the natural human complement of functional D gene segments; and/or

Optionally wherein the immunoglobulin locus comprises more than the natural human complement of functional J gene segments.

In this way, a superhuman immunoglobulin gene repertoire is provided in a transgenic non-human vertebrate or vertebrate cell according to the invention.

The First Configuration Also Provides:—

A transgenic non-human vertebrate (e.g., a mouse or rat) or vertebrate cell (optionally an ES cell or antibody-producing cell) whose genome comprises a transgenic immunoglobulin locus comprising a plurality of human immunoglobulin gene segments operably connected upstream of a non-human vertebrate constant region for the production of a repertoire of chimaeric antibodies, or chimaeric light or heavy chains, having a non-human vertebrate constant region and a human variable region; wherein the transgenic locus comprises one or more human immunoglobulin V gene segments, one or more human J gene segments and optionally one or more human D gene segments, a first (optionally a V segment) of said gene segments and a second (optionally a V segment) of said gene segments being different and derived from the genomes of first and second human individuals respectively, wherein the individuals are different; and optionally not related;

optionally wherein the immunoglobulin locus comprises more than the natural human complement of functional V gene segments; and/or optionally wherein the immunoglobulin locus comprises more than the natural human complement of functional D gene segments; and/or optionally wherein the immunoglobulin locus comprises more than the natural human complement of functional J gene segments.

In this way, a superhuman immunoglobulin gene repertoire is provided in a transgenic non-human vertebrate or vertebrate cell according to the invention.

The First Configuration Also Provides:—

A transgenic non-human vertebrate (e.g., a mouse or rat) or vertebrate cell (optionally an ES cell or antibody-producing cell) whose genome comprises first and second transgenic immunoglobulin loci, each locus comprising a plurality of human immunoglobulin gene segments operably connected upstream of a non-human vertebrate constant region for the production of a repertoire of chimaeric antibodies, or chimaeric light or heavy chains, having a non-human vertebrate constant region and a human variable region;

wherein (i) the first transgenic locus comprises one or more human immunoglobulin V gene segments, one or more human J gene segments and optionally one or more human D gene segments, (ii) the second transgenic locus comprises one or more human immunoglobulin V gene segments, one or more human J gene segments and optionally one or more human D gene segments; and (iii) wherein a first (optionally a V) gene segment of said first locus and a second (optionally a V) gene segment of said second gene locus are different and derived from the genomes of first and second human individuals respectively, wherein the individuals are different; and optionally not related; optionally wherein the first and second loci are on different chromosomes (optionally chromosomes with the same chromosome number) in said genome; optionally wherein each immunoglobulin locus comprises more than the natural human complement of functional V gene segments; and/or optionally wherein each immunoglobulin locus comprises more than the natural human complement of functional D gene segments; and/or optionally wherein each immunoglobulin locus comprises more than the natural human complement of functional J gene segments.

In this way, a superhuman immunoglobulin gene repertoire is provided in a transgenic non-human vertebrate or vertebrate cell according to the invention.

In these embodiments of the first configuration, the immunoglobulin gene segments are optionally as described for the third configuration.

In these embodiments of the first configuration, the genome optionally comprises a third immunoglobulin gene segment (optionally a V segment), the third gene segment being derived from a human individual that is different from the individual from which the first (and optionally also the second) gene segment is derived; optionally wherein the first, second and third gene segments are polymorphic variants of a human immunoglobulin gene segment (e.g., VH1-69—see the examples for further description).

In these embodiments of the first configuration, the genome of the vertebrate or cell is optionally homozygous for the first, second and optional third gene segment, wherein a copy of the first, second and optional third gene segments are provided together on the same chromosome operably connected upstream of a common non-human vertebrate constant region.

For example, each first, second and optional third gene segment is a V gene segment.

In one example, the library of the invention is provided by a collection of non-human vertebrates (optionally a collection of rodents, mice or rats); optionally, wherein a first member of said collection produces said first antibody but not said second antibody, and a second member of the collection produces said second antibody (but optionally not said first antibody). It is therefore contemplated to make non-human vertebrates where different human genomes have been used as a source for building the transgenic loci in the vertebrates. For example, a first vertebrate comprises a transgenic heavy chain locus having gene segments only from a first (and optionally a second) human population or individual; a second vertebrate comprises a transgenic heavy chain locus having gene segments only from a third (and optionally a fourth) human population or individual; and optionally third and more vertebrates can be built similarly based on unique or overlapping human population genomes. However, when provided as a mixed population of transgenic vertebrates, the mixed population provides a collective pool of human immunoglobulin genes that is greater than found in a natural human repertoire. This is useful to extend the antibody and gene sequence space beyond those possible with prior transgenic mice and rats bearing human immunoglobulin loci. As explained above, these have been based on a single human genome.

In one embodiment, the collection of non-human vertebrates bear human immunoglobulin genes confined to human populations that are together grouped under the same population genus “(a)” mentioned above. This provides for a gene repertoire that is biased to producing human antibody variable regions prevalent in the population genus (a) and thus useful for generating antibody therapeutics/prophylactics for members of said population. Alternatively, where gene segments from different human populations are provided in a single transgene according to the invention (not necessarily in a collection of vertebrates), the different human populations are for example together grouped under the same population genus “(a)” mentioned above.

The invention also provides a repertoire of antibodies expressed from a library of cells according to the invention.

In the non-human vertebrate or cell of any configuration of the invention, the constant region of the transgenic locus is, in one example, an endogenous constant region of said vertebrate (e.g., endogenous mouse or rat constant region, e.g., from the same strain of mouse or rat as the non-human vertebrate itself).

The invention also provides a method of constructing a cell (e.g., an ES cell) according to the invention, the method comprising

-   (a) identifying functional V and J (and optionally D) gene segments     of the genome sequence of a (or said) first human individual; -   (b) identifying one or more functional V and/or D and/or J gene     segments of the genome sequence of a (or said) second human     individual, wherein these additional gene segments are not found in     the genome sequence of the first individual; -   (c) and constructing a transgenic immunoglobulin locus in the cell,     wherein the gene segments of (a) and (b) are provided in the locus     operably connected upstream of a constant region.

Optionally the cell comprises a heavy chain locus constructed according to steps (a) to (c) and/or a light chain locus (kappa and/or lambda loci) constructed according to steps (a) to (c).

Optionally the cell is homozygous for the or each transgenic locus; optionally wherein antibody expression from loci endogenous to said cell has been inactivated. This is useful for confining the functional antibody gene repertoire, and thus antibody production, to antibodies bearing human variable regions.

Optionally the gene segment(s) in step (b) are identified from an immunoglobulin gene database selected from the 1000 Genomes, Ensembl, Genbank and IMGT databases.

Optionally the first and second human individuals are members of first and second ethnic populations respectively, wherein the populations are different, optionally wherein the human immunoglobulin gene segment derived from the genome sequence of the second individual is low-frequency (optionally rare) within the second ethnic population.

The invention also provides a method of making a transgenic non-human vertebrate (e.g., a mouse or rat), the method comprising

-   (a) constructing an ES cell (e.g., a mouse C57BL/6N, C57BL/6J, 129S5     or 129Sv strain ES cell) by carrying out the method above; -   (b) injecting the ES cell into a donor non-human vertebrate     blastocyst (e.g., a mouse C57BL/6N, C57BU6J, 129S5 or 129Sv strain     blastocyst); -   (c) implanting the blastocyst into a foster non-human vertebrate     mother (e.g., a C57BL/6N, C57BL/6J, 129S5 or 129Sv strain mouse);     and -   (d) obtaining a child from said mother, wherein the child genome     comprises a transgenic immunoglobulin locus.

The invention provides a transgenic non-human vertebrate (e.g., a mouse or rat) made by the method or a progeny thereof. The invention also provides a population of such non-human vertebrates.

Microinjection of ES cells into blastocysts and generation of transgenic mice therafter are conventional practices in the state of the art, and the skilled person is aware of techniques useful to effect this. C57BL/6N, C57BL/6J, 129S5 or 129Sv mouse strains and ES cells are readily and publicly available.

The invention also provides a method of isolating an antibody that binds a predetermined antigen (e.g., a bacterial or viral pathogen antigen), the method comprising

-   (a) providing a vertebrate (optionally a mouse or rat) according to     the invention; -   (b) immunizing (e.g., using a standard prime-boost method) said     vertebrate with said antigen (optionally wherein the antigen is an     antigen of an infectious disease pathogen); -   (c) removing B lymphocytes from the vertebrate and selecting one or     more B lymphocytes expressing antibodies that bind to the antigen; -   (d) optionally immortalizing said selected B lymphocytes or progeny     thereof, optionally by producing hybridomas therefrom; and -   (e) isolating an antibody (e.g., and IgG-type antibody) expressed by     the B lymphocytes; and -   (f) optionally producing a derivative or variant of the antibody.

This method optionally further comprises after step (e) the step of isolating from said B lymphocytes nucleic acid encoding said antibody that binds said antigen; optionally exchanging the heavy chain constant region nucleotide sequence of the antibody with a nucleotide sequence encoding a human or humanized heavy chain constant region and optionally affinity maturing the variable region of said antibody; and optionally inserting said nucleic acid into an expression vector and optionally a host.

Bioinformatics Analysis & Selection of Immunoglobulin Gene Segments

See also the discussion on variation analysis above.

The skilled person will know of sources of human antibody gene sequences, such as IMGT (www.imgt.org) GenBank (www.ncbi.nlm.nih.gov/genbank) Bioinformatics tools for database manipulation are also readily available and known to the skilled person, e.g., as publicly available from the 1000 Genomes Project/EBI (www.1000genomes.org)

As a source of antibody gene segment sequences, the skilled person will also be aware of e following available databases and resources (including updates thereof):—

-   1.1. The Kabat Database (G. Johnson and T. T.Wu, 2002;     http://www.kabatdatabase.com). Created by E. A. Kabat and T. T. Wu     in 1966, the Kabat database publishes aligned sequences of     antibodies, T-cell receptors, major histocompatibility complex (MHC)     class I and II molecules, and other proteins of immunological     interest. A searchable interface is provided by the SeqhuntII tool,     and a range of utilities is available for sequence alignment,     sequence subgroup classification, and the generation of variability     plots. See also Kabat, E. A., Wu, T. T., Perry, H., Gottesman, K.,     and Foeller, C. (1991) Sequences of Proteins of Immunological     Interest, 5th ed., NIH Publication No. 91-3242,Bethesda, Md., which     is incorporated herein by reference, in particular with reference to     human gene segments for use in the present invention. -   1.2. KabatMan (A. C. R. Martin, 2002;     http://www.bioinforg.uk/abs/simkab.html). This is a web interface to     make simple queries to the Kabat sequence database. -   1.3. IMGT, the International ImMunoGeneTics Information System®;     M.-P. Lefranc, 2002; http://imgt.cines.fr). IMGT is an integrated     information system that specializes in antibodies, T cell receptors,     and MHC molecules of all vertebrate species. It provides a common     portal to standardized data that include nucleotide and protein     sequences, oligonucleotide primers, gene maps, genetic     polymorphisms, specificities, and two-dimensional (2D) and     three-dimensional (3D) structures. IMGT includes three sequence     databases (IMGT/LIGM-DB, IMGT/MHC-DB, IMGT/PRIMERDB), one genome     database (IMGT/GENE-DB), one 3D structure database     (IMCT/3Dstructure-DB), and a range of web resources (“IMGT     Marie-Paule page”) and interactive tools. -   1.4. V-BASE (I. M. Tomlinson, 2002;     http://www.mrc-cpe.cam.ac.uk/vbase). V-BASE is a comprehensive     directory of all human antibody germline variable region sequences     compiled from more than one thousand published sequences. It     includes a version of the alignment software DNAPLOT (developed by     Hans-Helmar Althaus and Werner Muller) that allows the assignment of     rearranged antibody V genes to their closest germline gene segments. -   1.5. Antibodies-Structure and Sequence (A. C. R. Martin, 2002;     http://www.bioinforg.uk/abs). This page summarizes useful     information on antibody structure and sequence. It provides a query     interface to the Kabat antibody sequence data, general information     on antibodies, crystal structures, and links to other     antibody-related information. It also distributes an automated     summary of all antibody structures deposited in the Protein Databank     (PDB). Of particular interest is a thorough description and     comparison of the various numbering schemes for antibody variable     regions. -   1.6. AAAAA—AHo's Amazing Atlas of Antibody Anatomy (A. Honegger,     2001; http://www.unizh.ch/˜antibody). This resource includes tools     for structural analysis, modeling, and engineering. It adopts a     unifying scheme for comprehensive structural alignment of antibody     and T-cell-receptor sequences, and includes Excel macros for     antibody analysis and graphical representation. -   1.7. WAM—Web Antibody Modeling (N. Whitelegg and A. R. Rees, 2001;     http://antibody.bath.ac.uk). Hosted by the Centre for Protein     Analysis and Design at the University of Bath, United Kingdom. Based     on the AbM package (formerly marketed by Oxford Molecular) to     construct 3D models of antibody Fv sequences using a combination of     established theoretical methods, this site also includes the latest     antibody structural information. -   1.8. Mike's Immunoglobulin Structure/Function Page (M. R. Clark,     2001; http://www.path.cam.ac.uk/˜mrc7/mikeimages.html) These pages     provide educational materials on immunoglobulin structure and     function, and are illustrated by many colour images, models, and     animations. Additional information is available on antibody     humanization and Mike Clark's Therapeutic Antibody Human Homology     Project, which aims to correlate clinical efficacy and     anti-immunoglobulin responses with variable region sequences of     therapeutic antibodies. -   1.9. The Antibody Resource Page (The Antibody Resource Page, 2000;     http://www.antibodyresource.com). This site describes itself as the     “complete guide to antibody research and suppliers.” Links to amino     acid sequencing tools, nucleotide antibody sequencing tools, and     hybridoma/cell-culture databases are provided. -   1.9. Humanization bYDesign (J. Saldanha, 2000;     http://people.cryst.bbk.ac.uk/˜ubcg07s). This resource provides an     overview on antibody humanization technology. The most useful     feature is a searchable database (by sequence and text) of more than     40 published humanized antibodies including information on design     issues, framework choice, framework back-mutations, and binding     affinity of the humanized constructs.

See also Antibody Engineering Methods and Protocols, Ed. Benny K C Lo, Methods in Molecular Biology™, Human Press. Also at http://www.blogsua.com/odf/antibody-engineering-methods-and-protocolsantibody-engineering-methods-and-protocols.pdf

As a source of genomic sequence variation data, the skilled person will also be aware of the following available databases and resources (including updates thereof):—

-   1. HapMap (The International HapMap Consortium. 2003;     http://hapmap.ncbi.nlm.nih.gov/index.html.en). The HapMap Project is     an international project that aims to compare the genetic sequences     of different individuals to identify chromosomal regions containing     shared genetic variants. The HapMap www site provides tools to     identify chromosomal regions and the variant therein, with options     to drill down to population level frequency data. -   2. 1000 Genomes (The 1000 Genomes Project Consortium 2010;     http://www.1000genomes.org/). This resource provides complete     genomic sequence for 2500 unidentified individuals from one of 25     distinct population groups, with the aim of identifying genomic     variants of >1%. The site provides the ability to interrogate data     utilizing online tools (e.g. ‘Variation Pattern Finder’) and to     download variant data for individual population groups. -   3. Japanese SNP Database (H. Haga et al. 2002;     http://snp.ims.u-tokyo.ac.jp/index.html). Based on a study     identifying 190,562 human genetic variants this site catalogues     genomic variants with useful features for searching and summarizing     data.

It is possible to identify variants in immunoglobulin genes classed as low-frequency or rare variants that segregate with specific human ethnic populations. For the purpose of this analysis, a low-frequency immunoglobulin gene segment is classed as one with ‘Minor Allele Frequency’ (MAF) (cumulative frequency) of between 0.5%-5%, rare variants are those classed as having a MAF of less than 0.5% in a particular human population.

The following bioinformatics protocol is envisaged to identify human immunoglobulin gene segments for use in the present invention:

-   (a) Identify one or more genomic regions containing gene segments of     interest (‘target genomic regions’) and calculate the genomic     coordinates, using coordinates that match the sequence assembly     build used by either the 1000 Genomes project or International     HapMap project (or another selected human gene database of choice). -   (b) Identify genomic variants mapped to the genomic regions     previously identified in (a). Retrieve variant frequencies for     variants for each super population and preferably sub-population     where such data is available. Tools readily available on the HapMap     WWW site and the VWC tools for the 1000Genomes Project are useful     for this step. -   (c) Filter list of genomic variants from target genomic regions to     contain only variants classed as either ‘Non-synonymous’ single     nucleotide polymorphisms (SNPs) or genomic ‘insertions or     defections’ (indels). Filter further to include those that are     present in exonic sequences only. -   (d) Correlate population frequency data for each of the identified     variants for each of the super populations (for example ‘European     Ancestry’, ‘East Asian ancestry’, ‘West African ancestry’,     ‘Americas’, and ‘South Asian ancestry’) to identify those variants     that segregate with less than two super-populations. Further     correlate all identified variants with each of the sub-populations     (for example, ‘European ancestry’ super-population might be     subdivided into groups such as ‘CEU—Utah residents with Northern or     Western European ancestry’, ‘TSI Toscani in Italia’ and ‘British     from England and Scotland’) and produce a second score for rarity of     variants in within a super-population. -   (e) Collect one or more gene segments that show segregation to     specific sub-populations for construction of synthetic loci     according to the invention.

In one embodiment throughout the present text, “germline” refers to the canonical germline gene segment sequence.

By detailed analysis of the 1000 Genomes database, the inventors have devised a collection of candidate polymorphic antibody gene segment variants, e.g., human variant JH gene segments (e.g., see Example 4), that can be built into the design of transgenic heavy chain loci in mice for expressing increasingly diverse and new, synthetic repertoires of human variable regions. To this end, the invention provides the following embodiments.

The Present Invention Provides in a Fourth Configuration— Selection of Human JH6*02 Variant Transgenic IgH Loci, Non-Human Vertebrates, Cells & Antibodies Based on Human JH6*02

As explained above, in designing transgenic Ig heavy chain loci the present inventors have considered the huge amount of data available from the 1000 Genomes project (see www.1000genomes.org) that analyses gene distributions amongst many human populations, and in particular data on Ig gene segments. The inventors were also aware of human gene segments disclosed in the IMGT database (see www.imgt.org) and in Ensembl (see www.ensembl.org). The inventors needed to make choices about which human gene segments to include amongst the large number of human gene segments presented in these databases and the other sources of human Ig gene segment information known in the art, including those other databases disclosed herein. When choosing human JH gene segments, the inventors were aware that human JH6 encodes a relatively long amino acid sequence, and thus the inventors thought it desirable to include this for increasing the chances of producing IgH chains with relatively long HCDR3 regions. Antibodies with long HCDR3 (at least 20 amino acids according to IMGT nomenclature) have been shown to neutralize a variety of pathogens effectively including HIV, Influenza virus, malaria and Africa trypanosomes. Reference is also made to naturally-occurring Camelid (e.g., llama or camel) heavy chain-only antibodies which bear long HCDR3s for reaching relatively inaccessible epitopes (see, e.g., EP0937140). Long HCDR3s can form unique stable subdomains with extended loop structure that towers above the antibody surface to confer fine specificity. In some cases, the long HCDR3 itself is sufficient for epitope binding and neutralization (Liu, L et al; Journal of Virology. 2011. 85: 8467-8476, incorporated herein by reference). The unique structure of the long HCDR3 allows it to bind to cognate epitopes within inaccessible structure or extensive glycosylation on a pathogen surface. In human peripheral blood, there is around 3.5% of naive B antibodies or 1.9% of memory B IgG antibodies containing the HCDR3s with lengths of more than 24 amino acids (PLoS One. 2012; 7(5):e36750. Epub 2012 May 9; “Human peripheral blood antibodies with long HCDR3s are established primarily at original recombination using a limited subset of germline genes”; Briney BS e al, incorporated herein by reference) (FIG. 1). The usage analysis indicates that these antibodies have the preference to use human JH6 with human D2-2, D3-3 or D2-15 (Brinley, B S et al, FIGS. 2-5). See also PLoS One. 2011 Mar. 30; 6(3):e16857; Comparison of antibody repertoires produced by HIV-1 infection, other chronic and acute infections, and systemic autoimmune disease”; Breden F et al, incorporated herein by reference. Around 20% of all HCDR3 of antibodies use JH6. However, in those antibodies with HCDR3 of more than 24 amino acids, 70% use JH6 (Brinley, B S et al, FIG. 2).

There is a need in the art for genetically modified non-human vertebrates and cells that can make antibodies and heavy chains that have long human HCDR3s, as well as antibodies, chains and VH domains that can be selected from such vertebrates and cells wherein these can address target epitopes better accessed by long HCDR3s.

The inventors, therefore, chose in this configuration of the invention to include a human JH6 gene segment as a mandatory human gene segment in their IgH locus design. Several different naturally-occurring human JH6 variants are known (e.g., JH6*01 to *04 as well as others; IMGT nomenclature). The inventors considered this when deciding upon which human JH6 variant should be included in the transgenic IgH locus design. An alignment of some human JH6 variants is shown in FIG. 7 (from www.imgt.org; dashes indicate identical nucleotides; nucleotide changes versus the *01 variant are shown by underlined nucleotides and corresponding amino acid changes are shown by underlined amino acids; Genbank accession numbers (release 185.0) are shown prefixed by J, X, M or A). The inventors used sequencing of human genomic DNA samples, inspection of public IgH DNA databases as well as informed choices on the basis of variant sequences as means to arrive at a rational choice of which JH6 variant to use.

The 1000 Genomes database uses human JH6*03 as the reference sequence, which would be a possible choice for the skilled person wishing to construct a transgenic IgH locus. The inventors noticed (e.g., FIG. 7 herein) that position 6 in JH6*03 is a tyrosine (Y) encoded by a TAC codon, whereas some other naturally-occurring human variants have a glycine (G) encoded by a GGT codon (the glycine being present as a YYG motif, forming part of a larger YYGXDX motif). To understand the potential significance of this, the inventors carried out analysis of JH sequences from other vertebrate species. The inventors surprisingly noticed that YYG and YYGXDX motifs are conserved across many vertebrate species (see FIGS. 7 & 8). This suggested to the inventors, therefore, that preservation of this motif might be desirable, which could guide the choice of JH6 variant for use in the present invention.

Another pointer arose when the inventors considered the TAC codon versus the GGT codon encoding Y or G respectively. The inventors considered the impact of these nucleotide sequences on the action of activation-induced cytidine deaminase (AID). The inventors knew that activation-induced cytidine deaminase (AID) is believed to initiate Ig somatic hypermutation (SHM) in a multi-step mechanism and they addressed this activity when rationally designing the locus. AID catalyses the deamination of C to U in DNA, generating mutations at C bases. Cytidines located within hotspot motifs are preferentially deaminated. Certain motifs are hotspots for AID activity (DGYW, WRC, WRCY, WRCH, RGYW, AGY, TAC, WGCW, wherein W=A or T, Y=C or T, D=A, G or T, H=A or C or T, and R=A or G). The presence of a TAC codon encoding Y at position 6 in JH6*03 creates AID mutation hotspots (the cytidine being the substrate of AID), these hotspots being the underlined motifs in the previous sentence. The inventors considered the impact of this and in doing so they considered possible mutants created by AID activity at the cytidine. Reference is made to FIG. 9. The inventors noticed that a mutation at the third base of the TAC codon would yield 3 possible outcomes: Y, stop or stop. Thus, out of the three stop codons possible in the genetic code (the other being encoded by TGA—see FIG. 9), two of them would be provided by mutation of the cytidine in the TAC codon encoding position 6 in JH6*03. The inventors, therefore, considered that this might increase the chances of non-productive IgH variable region production in transgenic loci based on JH6*03. Moreover, the inventors noticed that provision of a GGT codon instead (as per the other human JH6 variants) seemed preferable since mutation of the third base would never yield a stop codon (see FIG. 9), and furthermore would retain coding, and thus conservation, of glycine at position 6, which the inventors also noticed was is in the YYG and YYGXDX motifs conserved across species.

Having decided against using JH6*03, the inventors needed to make a choice from other possible human variants. The MDV motif is at the C-terminus of HCDR3 based on human JH6, the adjacent framework 4 (FW4) starting with the WGQ motif (with reference to the sequence shown encoded by JH6*01; FIG. 7). In making their choices for locus design, the inventors wished to maximise conservation of this HCDR3/FW4 junction in product IgH chains and antibodies including these. The inventors believed this to be desirable for heavy chain variable domain functionality and conformation. The inventors thought that this might in some cases be desirable to minimise immunogenicity (suitable for human pharmaceutical use). Consistent with these considerations, the inventors wanted to make a choice that would minimise mutation around the HCDR3/FW4 junction as a result of SHM in vivo to conserve junction configuration. See Rogozin & Diaz; “Cutting Edge: DGYW/WRCH Is a Better Predictor of Mutability at G:C Bases in Ig Hypermutation Than the Widely Accepted RGYW/WRCY Motif and Probably Reflects a Two-Step Activation-Induced Cytidine Deaminase-Triggered Process”; Journal of Immunology; Mar. 15, 2004 vol. 172 no. 6 3382-3384. An example of a DGYW motif is GGCA. The inventors had this in mind when analysing the variant sequences.

With these considerations in mind, the inventors decided specifically to use human JH6*02 as the mandatory human JH6 for their IgH locus design. JH6*01 was rejected as the mandatory JH6 gene segment since the nucleotide sequence GGG CAA (encoding G and Q) contains a GGCA motif which is an AID recognition hotspot. The inventors realised that JH6*04 also contains such a motif due to the presence of the sequence GGC AAA encoding G and K (positions 11 and 12 respectively). The inventors also realised that the *02 variant has a C instead of a G that is in the *01 variant, the C desirably being a synonymous change (i.e., not changing the encoded amino acid sequence around the CDR3/FW4 junction) and also this does not provide a GGCA AID hotspot motif. The inventors, therefore, decided that the mandatory JH6 should have this C base and this too pointed them to using the human JH6*02 variant.

In one example of any configuration of the invention herein, the only JH6 species included in the locus or genome is human JH6*02.

The inventors obtained 9 anonymised DNA samples from cheek swabs of 9 consenting human adults. Sequencing was performed on IgH locus DNA to confirm natural JH6 variant usage. It was found that the genome of all 9 humans contained a JH6*02 variant gene segment. In 7 out of the 9 humans, the genome was homozygous for JH6*02 (i.e., each chromosome 14 had JH6*02 as its JH6 gene segment in the IgH locus). The inventors also inspected the publicly-available sequence information from the genomes of well-known scientists Craig Venter and Jim Watson. Both of these genomes contain JH6*02 too. This indicated to the inventors that this variant is common in humans.

So, the inventors made a choice of human JH6*02 on the basis of

-   (i) Containing the YYG and YYGXDX motifs that is conserved across     several vertebrate species; -   (ii) Provision of one less TAC codon (an AID hotspot that risks stop     codons) and a choice instead of a codon that preserves the YYG and     YYGXDX motifs; -   (iii) Avoidance of a GGCA AID hotspot in the region of the HCDR3/FW4     junction; and -   (iv) Common occurrence (and thus conservation and acceptability) in     humans of the JH6*02 variant.

This rationale was tested by the inventors in laboratory examples, in order to see if human JH6*02 could desirably participate in antibody gene segment recombination and heavy chain production in a foreign (non-human vertebrate) setting, and moreover to assess if long HCDR3s based on human JH6*02 could be produced in vivo (in naive and immunised settings) in such non-human systems. It was noted that in some non-human settings, such as a mouse, the YYG and YYGXDX motifs are not conserved, and thus the inventors decided that it was important to test whether or not JH6*02 (having the YYG and YYGXDX motifs) could function properly in such a foreign setting to participate in VDJ recombination and selection against antigen.

Thus, as explained further in the examples, the inventors constructed transgenic JH6*02-containing IgH loci in ES cells, generated transgenic non-human vertebrates from the ES cells (both naive and immunised with a range of different target antigen types), isolated antibodies and heavy chain sequences based on JH6*02 as well as B-cells expressing these and made hybridomas expressing antigen-specific antibodies that are based on the chosen JH6*02 variant. The inventors found that the JH6*02 variant was extensively used and could contribute to the production of HCDR3 of at least 20 amino acids in many different heavy chains (including antigen-specific heavy chains). The chosen variant was preferably used over other JH gene segments in all settings (naive, immunised and antigen-specific) for the production of HCDR3 of at least 20 amino acids.

Thus, the present invention provides an IgH locus including human JH6*02 (IMGT nomenclature) as a mandatory JH gene segment. In one embodiment, the locus comprises non-human vertebrate (e.g., mouse or rat) constant region gene segments downstream (i.e., 3′ of) the human JH6*02; and one or more VH gene segments (e.g., a plurality of human VH gene segments) and one or more D gene segments (e.g., a plurality of human D gene segments) upstream of (i.e., 5′ of) the human JH6*02. For example, the locus is comprised by a vector (e.g., a DNA vector, e.g., a yeast artificial chromosome (YAC), BAC or PAC). Such a vector (e.g., YAC) can be introduced into a non-human vertebrate (e.g., mouse or rat) cell using standard techniques (e.g., pronuclear injection) so that the locus is integrated into the cell genome for expression of IgH chains comprising at least one chain whose variable domain is a product of the recombination of human JH6*02 with a VH and a D gene segment.

In another example, the locus (e.g., with a completely human, rat or mouse constant region, or a human/mouse chimaeric constant region) can be provided in the genome of a non-human vertebrate (e.g., mouse or rat) cell. For example, the cell is an ES cell or an antibody-producing cell (e.g., an isolated B-cell, an iPS cell or a hybridoma).

In another example, the invention provides a non-human vertebrate (e.g., a mouse or a rat) comprising an IgH locus of the invention which comprises a human JH6*02 gene segment, wherein the locus can express an IgH chain whose variable domain is a product of the recombination of human JH6*02 with a VH and a D gene segment. As shown in the examples, the inventors have successfully produced such mice which produce such IgH chains with VH domains based on human JH6*02. The inventors isolated and sequenced IgH chains from the mice before (naive) and after (immunised) exposure to a range of target antigens and confirmed by comparison to IMGT IgH gene segment sequences that the isolated chains (and antibodies containing these) were produced based on JH6*02. Such chains were found in naive mice, as well as in antigen-specific antibodies from immunised mice. B-cells were isolated from immunised mice, wherein the B-cells express antibodies based on JH6*02 and hybridomas were generated from the B-cells, the hybridomas expressing antigen-specific antibodies based on JH6*02. The inventors, therefore, provided the locus, vertebrate, cell and hybridoma of the invention based on the use of human JH6*02 and showed that antibodies based on JH6*02 and B-cells expressing these can be successfully produced and isolated following immunisation of the vertebrates, corresponding hybridomas being a good source of antibodies whose VH domains are based on JH6*02, e.g. for administration to a patient, e.g., for human medicine. Furthermore, it was found possible to produce and isolated antigen-specific antibodies whose VH domains are based on JH6*02 and which had a relatively long HCDR3 (e.g., 20 amino acids).

Thus, the present invention provides embodiments as in the following clauses:—

-   1. A non-human vertebrate (optionally a mouse or a rat) or     vertebrate cell whose genome comprises an immunoglobulin heavy chain     locus comprising human gene segment JH6*02, one or more VH gene     segments and one or more D gene segments upstream of a constant     region; wherein the gene segments in the heavy chain locus are     operably linked to the constant region thereof so that the mouse is     capable of producing an antibody heavy chain produced by     recombination of the human JH6*02 with a D segment and a VH segment.     -   In another example, the invention provides     -   A non-human vertebrate (optionally a mouse or a rat) or         vertebrate cell whose genome comprises an immunoglobulin heavy         chain locus comprising one, more or all of human IGHV gene         segments selected from V3-21, V3-13, V3-7, V6-1, V1-8, V1-2,         V7-4-1, V1-3, V1-18, V4-4, V3-9, V3-23, V3-11 and V3-20 (e.g.,         one, more or all of V3-21*03, V3-13*01, V3-7*01, V6-1*01,         V1-8*01, V1-2*02, V7-4-1*01, V1-3*01, V1-18*01, V4-4*01, V3-9*01         and V3-23*04). These segments were found in naive repertoires to         be productive to produce HCDR3s of at least 20 amino acids in         length. In an embodiment, the locus comprises a human JH6, e.g.,         JH6*02.     -   The invention also provides a HCDR3, VH domain, antibody heavy         chain or antibody having a HCDR3 size of at least 20 amino         acids. Optionally, the HCDR3 or VH domain (or VH domain of the         heavy chain or antibody) comprises mouse AID-pattern somatic         hypermutations and/or mouse dTd-pattern mutations. This can be         provided, for example, wherein VH domain is produced in a mouse         comprising mouse AID and/or mouse TdT (e.g., endogenous AID or         TdT). See also Annu. Rev. Biochem. 2007. 76:1-22; Javier M. Di         Noia and Michael S. Neuberger, “Molecular Mechanisms of Antibody         Somatic Hypermutation” (in particular FIG. 1 and associated         discussion on AID hotspots in mouse); and Curr Opin Immunol.         1995 April; 7(2):248-54, “Somatic hypermutation”, Neuberger M S         and Milstein C (in particular, discussion on hotspots in mouse),         the disclosures of which are incorporated herein by reference.     -   These segments were found in naive repertoires to be productive         in recombination with human JH6*02 to produce HCDR3s of at least         20 amino acids in length.     -   In an example, the vertebrate is naive. In another embodiment,         the vertebrate instead is immunised with a target antigen.     -   In an example, the vertebrate or cell mentioned below is capable         of so producing an antibody heavy chain upon immunisation with a         target antigen. In an example, the vertebrate is an immunised         vertebrate that produces antibody heavy chains specific for a         target antigen and wherein the variable domains of the heavy         chains are the product of recombination between a VH, D and         JH6*02. For example, the D is selected from human D3-3, D2-15,         D3-9; D4-17; D3-10; D2-2; D5-24; D6-19; D3-22; D6-13; D5-12;         D1-26; D1-20; D5-18; D3-16; D2-21; D1-14; D7-27; D1-1; D6-25;         D2-14 and D4-23 (e.g., selected from D3-9*01; D4-17*01;         D3-10*01; D2-2*02; D5-24*01; D6-19*01; D3-22*01; D6-13*01;         D5-12*01; D1-26*01; D1-20*01; D5-18*01; D3-16*02; D2-21*02;         D1-14*01; D7-27*02; D1-1*01; D6-25*01; D2-15*01; and D4-23*01).         For example, the D is human D3-9 or D3-10. In an example, the         HCDR3 length is at least 20 amino acids (e.g., 20, 21, 23 or         24).     -   In an example of the vertebrate or cell, the genome comprises         additional human JH gene segments (e.g., JH2, 3, 4 and 5 gene         segments).     -   In an example of the vertebrate or cell, the genome comprises an         immunoglobulin light chain locus comprising one or more human V         gene segments and one or more human J gene segments upstream of         a constant region (e.g., a human or a mouse lambda or kappa         constant region).     -   For rearrangement and expression of heavy chains, the locus         comprises control elements, such as an Ê and Ŝ between the J         gene segment(s) and the constant region as is known by the         skilled person. In one example, a mouse Ê and Ŝ is included in         the heavy chain locus between the JH6*02 and the constant region         (i.e., in 5′ to 3′ order the locus comprises the JH6*02, Ê and Ŝ         and constant region). In an example, the Ê and Ŝ are Ê and Ŝ of         a mouse 129-derived     -   genome (e.g., a 129Sv-derived genome, e.g., 129Sv/EV (such as         129S7Sv/Ev (such as from AB2.1 or AB2.2 cells obtainable from         Baylor College of Medicine, Texas, USA) or 129S6Sv/Ev))); in         another example, the Ê and Ŝ are Ê and Ŝ of a mouse         C57BL/6-derived genome. In this respect, the locus can be         constructed in the IgH locus of the genome of a cell selected         from AB2.1, AB2.2, VGF1, CJ7 and FH14. VGF1 cells were         established and described in Auerbach W, Dunmore J H,         Fairchild-Huntress V, et al; Establishment and chimera analysis         of 129/SvEv- and C57BU6-derived mouse embryonic stem cell lines.         Biotechniques 2000; 29:1024-8, 30, 32, incorporated herein by         reference.     -   Additionally or alternatively, the constant region (or at least         a Ĉ or Ĉ and gamma constant regions thereof) is a constant         region (or Ĉ or Ĉ and gamma constant regions thereof) is of a         genome described in the paragraph immediately above.     -   A suitable source of JH6*02 and other human DNA sequences will         be readily apparent to the skilled person. For example, it is         possible to collect a DNA sample from a consenting human donor         (e.g., a cheek swab sample as per the Example herein) from which         can be obtained suitable DNA sequences for use in constructing a         locus of the invention. Other sources of human DNA are         commercially available, as will be known to the skilled person.         Alternatively, the skilled person is able to construct gene         segment sequence by referring to one or more databases of human         Ig gene segment sequences disclosed herein. -   2. The vertebrate of clause 1, wherein the vertebrate has been     immunised with a target antigen and wherein the variable domain of     the heavy chain is the product of recombination between a VH, D and     JH6*02 and wherein the HCDR3 length is at least 20 amino acids     (e.g., 20, 21, 23 or 24).     -   Optionally, the immunised vertebrate produces an antibody heavy         chain specific for a target antigen and wherein the variable         domain of the heavy chain is the product of recombination         between a VH, D and JH6*02 and wherein the HCDR3 length is at         least 20 amino acids (e.g., 20, 21, 23 or 24). -   3. A non-human vertebrate cell (optionally a mouse cell or a rat     cell) whose genome comprises an immunoglobulin heavy chain locus     comprising human gene segment JH6*02, one or more VH gene segments     and one or more D gene segments upstream of a constant region;     wherein the gene segments in the heavy chain locus are operably     linked to the constant region thereof for producing (e.g., in a     subsequent progeny cell) an antibody heavy chain produced by     recombination of the human JH6*02 with a D segment and a VH segment. -   4. The cell of clause 3, which is an ES cell capable of     differentiation into a progeny antibody-producing cell that     expresses said heavy chain. -   5. The vertebrate or cell of any preceding clause, wherein the heavy     chain locus comprises a human JH6*02 recombination signal sequence     (RSS) operably connected 5′ to the JH6*02 gene segment.     -   For example, the native RSS-JH6*02 sequence can be used to         advantageously maintain the natural pairing between RSS and         theis JH gene segment. In this respect, the following sequence         is used:—     -   ggtttttgtggggtgaggatggacattctgccattgtgattactactactactacggtatggacgtctggggccaagggaccac         ggtcaccg tctcctcag (SEQ ID NO: 238)     -   RSSs have a common architecture: 9mer (e.g., first underlined         sequence above) followed by a 22 bp spacer and then a 7mer         (e.g., second underlined sequence above). Spacers are 23 bp+/−1         normally, while the 9 and 7mer are more conserved. -   6. The vertebrate or cell of clause 5, wherein the RSS is SEQ ID NO:     238 or a sequence having an identical 9mer and 7mer sequence     flanking a sequence that is at least 70% identical to the 22mer     sequence of SEQ ID NO: 238. -   7. The vertebrate or cell of clause 6, wherein the RSS and JH6*02     are provided as SEQ ID NO: 237. -   8. The vertebrate or cell of any preceding clause, wherein the     JH6*02 is the only JH6-type gene segment in the genome. -   9. The vertebrate or cell of any preceding clause, wherein the     JH6*02 is the closest JH gene segment to the constant region in the     locus. -   10. The vertebrate or cell of any preceding clause, wherein the     locus comprises one, more or all human D gene segments D3-9; D4-17;     D3-10; D2-2; D5-24; D6-19; D3-22; D6-13; D5-12; D1-26; D1-20; D5-18;     D3-16; D2-21; D1-14; D7-27; D1-1; D6-25; D2-14; and D4-23.     -   For example, the locus comprises one, more or all of human D         gene segments D3-9*01; D4-17*01; D3-10*01; D2-2*02; D5-24*01;         D6-19*01; D3-22*01; D6-13*01; D5-12*01; D1-26*01; D1-20*01;         D5-18*01; D3-16*02; D2-21*02; D1-14*01; D7-27*02; D1-1*01;         D6-25*01; D2-15*01; and D4-23*01. -   11. The vertebrate or cell of clause 10, wherein the locus comprises     one, more or all human D gene segments D3-9, D3-10, D6-19, D4-17,     D6-13, D3-22, D2-2, D2-25 and D3-3.     -   These D segments were found to be productive in recombination         with human JH6*02 to produce HCDR3s of at least 20 amino acids         in length.     -   In an example, the locus comprises one, more or all human D gene         segments D3-9, D3-10, D6-19, D4-17, D6-13 and D3-22 (for example         one, more or all of D3-9*01, D3-10*01, D6-19*01, D4-17*01,         D6-13*01 and D3-22*01). These D segments were found in naive         repertoires to be productive in recombination with human JH6*02         to produce HCDR3s of at least 20 amino acids in length.     -   In an example, the locus comprises one, more or all human D gene         segments D3-10, D6-19 and D1-26 (for example, one, more or all         of D3-10*01, D6-19*01 and D1-26*01). These D segments were found         in immunised repertoires to be productive in recombination with         human JH6*02 to produce HCDR3s of at least 20 amino acids in         length.     -   In an example, the locus comprises one, more or all human D gene         segments D3-9 and D3-10 (for example, one, more or all of         D3-9*01 and D3-10*01). These D segments were found in         antigen-specific repertoires to be productive in recombination         with human JH6*02 to produce HCDR3s of at least 20 amino acids         in length. -   12. The vertebrate or cell of any preceding clause, wherein the     locus comprises a plurality of human D gene segments and the JH6*02     is in human germline configuration with respect to the 3′-most human     D gene segment (or all of the human D segments comprised by the     locus).     -   In an example, the 3′-most D gene segment is D7-27. In an         example, the locus comprises all of human D gene segments from         D1-1 to D7-27 as present in a germline human IgH locus (e.g., as         shown in the IMGT database).     -   Alternatively or additionally, the JH6*02 is in human germline         configuration with respect to one, more or all of the Ê Ŝ and         constant region (e.g., Cu) -   13. The vertebrate or cell of any preceding clause, wherein the     locus comprises one, more or all of IGHV gene segments selected from     V3-21, V3-13, V3-7, V6-1, V1-8, V1-2, V7-4-1, V1-3, V1-18, V4-4,     V3-9, V3-23, V3-11 and V3-20.     -   In an example, the locus comprises one, more or all human IGHV         gene segments V3-21, V3-13, V3-7, V6-1, V1-8, V1-2, V7-4-1,         V1-3, V1-18, V4-4, V3-9, V3-23 (for example, one, more or all of         V3-21*03, V3-13*01, V3-7*01, V6-1*01, V1-8*01, V1-2*02,         V7-4-1*01, V1-3*01, V1-18*01, V4-4*01, V3-9*01 and V3-23*04).         These segments were found in naive repertoires to be productive         in recombination with human JH6*02 to produce HCDR3s of at least         20 amino acids in length.     -   In an example, the locus comprises one, more or all human IGHV         gene segments V3-7, V3-11 and V4-4 (for example, one, more or         all of V3-7*01, V3-11*01 and V4-4*02). These segments were found         in immunised repertoires to be productive in recombination with         human JH6*02 to produce HCDR3s of at least 20 amino acids in         length.     -   In an example, the locus comprises one, more or all human IGHV         gene segments V4-4, V1-8, V3-9, V3-11 and V3-20 (for example,         one, more or all of V4-4*02, V1-8*01, V3-9*01, V3-11*01 and         V3-20 (e.g., *d01). These segments were found in         antigen-specific repertoires to be productive in recombination         with human JH6*02 to produce HCDR3s of at least 20 amino acids         in length. -   14. The vertebrate or cell of any preceding clause, wherein the     locus comprises one, more or all of human D3-9*01, D3-10*01,     D6-19*01, D6-13*01, D1-26*01, IGHV1-8*01, IGHV4-61*01, IGHV6-1*01,     IGHV4-4*02, IGHV1-3*01, IGHV3-66*03, IGHV3-7*01 and IGHV3-9*01.     -   These are gene segments that very frequently combine with JH6*02         to produce productive heavy chains and antibodies.     -   For example, the locus comprises one, more or all of human         IGHV1-8*01, D3-9*01 and D3-10*01. These gene segments were         productive with JH6*02 to produce HCDR3s of at least 20 amino         acids in more than 10 antibodies. -   15. An antibody-producing cell (e.g., a B-cell) that is a progeny of     the cell of any one of clauses 3 to 14, wherein the     antibody-producing cell comprises a heavy chain locus comprising a     rearranged variable region produced by recombination of human JH6*02     with a D segment and a VH segment (e.g., JH6*02 with human VH3-11     (e.g., VH3-11*01) and D3-9; VH3-20 (e.g., VH3-20*01) and D3-10;     VH4-4 (e.g., VH4-4*02) and D3-10; VH3-9 (e.g., VH3-9*01) and D3-10;     or VH1-8 (e.g., VH1-8*01) and D310).     -   Such a variable region would be the product of in vivo somatic         hypermutation in a non-human vertebrate or cell of the         invention. -   16. The cell of clause 15, which is a B-cell or hybridoma that     expresses a target antigen-specific antibody comprising a heavy     chain that comprises a rearranged variable region produced by     recombination of human JH6*02 with a D segment and a VH segment     (e.g., JH6*02 with human VH3-11 (e.g., VH3-11*01) and D3-9; VH3-20     (e.g., VH3-20*01) and D3-10; VH4-4 (e.g., VH4-4*02) and D3-10; VH3-9     (e.g., VH3-9*01) and D3-10; or VH1-8 (e.g., VH1-8*01) and D310).     -   Such a variable region would be the product of in vivo somatic         hypermutation in a non-human vertebrate or cell of the invention -   17. The vertebrate or cell of any preceding clause, wherein the     antibody heavy chain specifically binds a target antigen. -   18. The vertebrate or cell of any preceding clause, wherein the     antibody heavy chain has a HCDR3 length of at least 20 amino acids.     -   Optionally, the HCDR3 length is at least 21, 22, 23, 24, 25, 26,         27, 28, 29 or 30 amino acids. Additionally, in one example the         length is no more than 35, 34, 33, 32 or 31 amino acids. For         example, the HCDR3 length is 20, 21, 22, 23 or 24 amino acids. -   19. The vertebrate or cell of any preceding clause, wherein the     antibody heavy chain is a product of the recombination of JH6*02     with a human VH gene segment recited in clause 13 or 14 and/or a D     gene segment recited in clause 10, 11 or 14. -   20. The vertebrate or cell of any preceding clause, wherein all     endogenous non-human vertebrate heavy chain variable region gene     segments have been inactivated in the genome (E.g., by gene segment     deletion or inversion). -   21. The vertebrate or cell of any preceding clause, wherein the     genome is homozygous for said heavy chain locus. -   22. A heavy chain (e.g., comprised by an antibody) isolated from a     vertebrate of any one of clauses 1, 2, 5 to 14 and 17 to 21 wherein     the heavy chain comprises a HCDR3 of at least 20 amino acids. -   23. The heavy chain of clause 22, wherein the HCDR3 is the product     of recombination of human JH6*02 with a human VH gene segment     recited in clause 13 or 14 and/or a D gene segment recited in clause     10, 11 or 14.     -   In an example, the heavy chain is chimaeric where the C region         is non-human. In an example, the heavy chain is human where the         C region is human. -   24. A heavy chain (e.g., comprised by an antibody) whose VH variable     domain is identical to the VH variable domain of the heavy chain of     clause 22 or 23, and which comprises a human constant region or a     human-mouse chimaeric constant region (e.g., CH1 is human and the     other constant domains are mouse). -   25. The heavy chain of clause 22, 23 or 24, whose VH variable domain     is specific for a target antigen. -   26. A method for producing a heavy chain, VH domain or an antibody     specific to a target antigen, the method comprising immunizing a     non-human vertebrate according to any one of clauses 1, 2, 5 to 14     and 17 to 21 with the antigen and isolating the heavy chain, VH     domain or an antibody specific to a target antigen or a cell     producing the heavy chain, VH domain or an antibody, wherein the     heavy chain, VH domain or an antibody comprises a HCDR3 that is     derived from the recombination of human JH6*02 with a VH gene     segment and a D gene segment. -   27. A method for producing a human heavy chain or antibody     comprising carrying out the method of clause 26, wherein the     constant region of the locus is a non-human vertebrate (e.g., mouse     or rat) constant region, and then replacing the non-human constant     region of the isolated heavy chain or antibody with a human constant     region (e.g., by engineering of the nucleic acid encoding the     antibody). -   28. A heavy chain, VH domain or an antibody produced by the method     of clause 26 or 27. Optionally the HCDR3 length is at least 20 amino     acids as herein described. -   29. A B-cell or hybridoma expressing a heavy chain VH domain that is     identical to the VH domain of the heavy chain of clause 22, 23 or     28. -   30. A nucleic acid encoding the VH domain of the heavy chain of     clause 22, 23 or 28, or encoding the heavy chain of clause 22, 23,     24, 25 or 28. -   31. A vector (e.g., a CHO cell or HEK293 cell vector) comprising the     nucleic acid of clause 30; optionally wherein the vector is in a     host cell (e.g., a CHO cell or HEK293 cell). -   32. A pharmaceutical composition comprising the antibody, heavy     chain or VH domain (e.g., comprised by an antibody) of any one of     clauses 22 to 25 and 28, together with a pharmaceutically-acceptable     excipient, diluent or a medicament (e.g., a further antigen-specific     variable domain, heavy chain or antibody). -   33. The antibody, heavy chain or VH domain (e.g., comprised by an     antibody) of any one of clauses 22 to 25 and 28 for use in medicine     (e.g., human medicine).

For example, the locus comprises the following human VH gene segments

IGHV6-1 IGHV3-7 IGHV1-8 IGHV3-9 IGHV3-11 IGHV3-13 IGHV1-18 IGHV3-30 IGHV4-31 IGHV4-39 IGHV4-59

Optionally also (i) and/or (ii)

(i)

IGHV1-2 IGHV2-5 and IGHV3-21

(ii)

IGHV1-2 IGHV2-5 IGHV3-21 IGHV1-24

For example, the locus comprises the following human VH gene segment variants

IGHV6-1*01 IGHV3-7′01 IGHV1-8′01 IGHV3-9′01 IGHV3-11*01 IGHV3-13*01 IGHV1-18′01 IGHV3-30′18 IGHV4-31′03 IGHV4-39*01 and IGHV4-59*01;

Optionally also (iii) or (iv)

(ii)

IGHV1-2*04 IGHV2-5*10 and IGHV3-21*03

(iv)

IGHV1-2′02 IGHV2-5′01 IGHV3-21′01 and IGHV1-24′01

For example, the locus comprises the following human JH gene segment variants

IGHJ2*01 IGHJ3*02 IGHJ4*02 IGHJ5*02 and IGHJ6*02

For example, the locus comprises the following human D gene segments

IGHD1-1 IGHD2-2 IGHD3-9 IGHD3-10 IGHD5-12 IGHD6-13 IGHD1-14 IGHD2-15 IGHD3-16 IGHD4-17 IGHD6-19 IGHD2-21 IGHD5-24 IGHD1-26 and IGHD7-27

and optionally also (v) or (vi) (v)

IGHD3-3

(vi)

IGHD3-3 IGHD4-4 IGHD5-5 IGHD6-6 IGHD1-7 IGHD2-8 and IGHD2-8 The Present Invention Provides in a Fifth Configuration— Constant Regions Tailored to Human Use & Antibody Humanization

Additional rational design and bioinformatics has led the inventors to realise that specific human constant region variants are conserved across many diverse human populations. The inventors realised that this opens up the possibility of making a choice to humanize antibodies, chains and variable domains by using such specific constant regions in products, rather than arbitrarily choosing the human constant region (or a synthetic version of a human constant region). This aspect of the invention also enables one to tailor antibody-based drugs to specific human ethnic populations, thereby more closely matching drug to patient (and thus disease setting) than has hitherto been performed. It can be a problem in the state of the art that antibodies are humanized with an arbitrary choice of human constant region (presumably derived from one (often unknown) ethnic population or non-naturally occurring) that does not function as well in patients of a different human ethnic population. This is important, since the constant region has the major role in providing antibody effector functions, e.g., for antibody recycling, cellular and complement recruitment and for cell killing.

As discussed further in WO2011066501, human IgG sub-types IgG1, IgG2, gG3 and IgG4 exhibit differential capacity to recruit immune functions, such as antibody-dependent cellular cytotoxicity (ADCC, e.g., IgG1 and IgG3), antibody-dependent cellular phagocytosis (ADCP, e.g., IgG1, IgG2, IgG3 and IgG4), and complement dependent cytotoxicity (CDC, e.g., IgG1, IgG3). Sub-type-specific engagement of such immune functions is based on selectivity for Fc receptors on distinct immune cells and the ability to bind C1q and activate the assembly of a membrane attack complex (MAC).

Among the various types, relative affinity for FcY receptors (e.g., FcYRI, FcYRIIa/b/c, FcYRIIIa/b) is high for IgG1 and IgG3, however, there is minimal affinity for IgG2 (restricted to the FcYRIIa 131H polymorphism), and IgG4 only has measurable affinity for FcYRI. Using comparative sequence analysis and co-crystal structures, the key contact residues for receptor binding have been mapped to the amino acid residues spanning the lower hinge and CH2 region. Using standard protein engineering techniques, some success in enhancing or reducing the affinity of an antibody preparation for Fc receptors and the C1q component of complement has been achieved.

Among the isotypes, IgG2 is least capable of binding the family of Fc receptors. Using IgG2 as the starting point, efforts have been made to find a mutant with diminished effector functions but which retains FcRn binding, prolonged stability, and low immunogenicity. Improved mutants of this nature may provide improved antibody therapeutics with retained safety. Human IgG1 therapeutic antibodies that bind to cell surface targets are able to engage effector cells that may mediate cell lysis of the target cell by antibody-dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). These mechanisms occur through interaction of the CH2 region of the antibody Fc domain to FcyR receptors on immune effector cells or with C1q, the first component of the complement cascade. Table 19 shows the activities of different human gamma sub-types. The skilled person may choose accordingly to promote or dampen-down activity depending upon the disease setting in humans of interest. For example, use of a human gamma-1 constant region is desirable when one wishes to isolated totally human heavy chains and antibodies that have relatively high complement activation activity by the classical pathway and FcYR1 recognition in human patients. See also Mol Immunol. 2003 December; 40(9):585-93; “Differential binding to human Fcgamma RIIe and Fcgamma RIIb receptors by human IgG wild type and mutant antibodies”; Armour K L et al, which is incorporated herein by reference.

IgG2 constant regions are well suited to producing antibodies and heavy chains according to the invention for binding to cytokines or soluble targets in humans, since IgG2 is essentially FcYRI,III-silent, FcYRIIa-active and has little Complement activity.

IgG1 constant regions have wide utility for human therapeutics, since IgG1 antibodies and heavy chains are FcYRI,II,III-active and have complement activity. This can be enhanced by using a human gamma-1 constant region that has been activated by engineering as is known in the art.

The work of the inventors has therefore identified a collection of human constant region of different isotypes from which an informed choice can be made when humanizing chimaeric antibody chains (or conjugating V domains, such as dAbs or Camelid VHH, to constant regions). The collection was identified on the basis of bioinformatics analysis of the 1000 Genomes database, the inventors selecting constant region variants that are frequently occurring across several human ethnic populations, as well as those that appear with relatively high frequency within individual populations (as assessed by the number of individuals whose genomes comprise the variant). By sorting through the myriad possible sequences on this basis, the inventors have provided a collection of human constant region variants that are naturally-occurring and which can be used when rationally designing

antibodies, heavy chains and other antibody-based formats that bear a human constant region. In particular, this is useful when humanizing chimaeric heavy chains to produce totally human chains in which both the variable and constant regions are human. This is useful for compatibility with human patients receiving antibody-based drugs.

To this end, the invention provides the following aspects:—

-   1. method of producing an antibody heavy chain, the method     comprising -   (a) providing an antigen-specific heavy chain variable domain (e.g.,     VH (such as a human VH or dAb) or VHH or a humanized heavy chain     variable domain); and -   (b) combining the variable domain with a human heavy chain constant     region to produce an antibody heavy chain comprising (in N- to     C-terminal direction) the variable domain and the constant region;     -   wherein     -   the human heavy chain constant region is an IGHAref, IGHA1a,         IGHA2a, IGHA2b, IGHG1ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a,         IGHG3b, IGHG4ref, IGHG4a, IGHDref, IGHEref, IGHMref, IGHMa or         IGHMb constant region.     -   Step (b) can be carried out, e.g., using recombinant DNA         technology using the corresponding nucleotide sequences.

For the constant region according to any aspect of this configuration, either genomic DNA or equivalent (i.e., having introns and exons and optionally also 5′ UTR sequences, e.g., with native or a non-native leader sequence) can be used for the constant region. For example, any of the “GENOMIC” sequences disclosed as SEQ ID NO: 365 onwards herein. Alternatively, an intron less sequence can be used, for example any of the “CDS” sequences disclosed as SEQ ID NO: 365 onwards herein (e.g., with native or a non-native leader sequence).

-   -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHAref         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHA1a         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHA2a         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHA2b         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is IGHG1 ref         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHG2ref         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHG2a         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHG3ref         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHG3a         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHG3b         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHG4ref         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHG4a         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHDref         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHEref         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHMref         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHMa         constant region.     -   Optionally for any aspect of this configuration of the         invention, the human heavy chain constant region is an IGHMb         constant region.     -   Optionally, a derivative (e.g., a mutant or conjugate) of the         heavy chain or an antibody containing the heavy chain is         produced. For example, a toxic payload can be conjugated (e.g.,         for oncology applications). For example, one or more mutations         can be introduced, as is known in the art, to inactivate or         enhance Fc effector function.

-   2. The method of aspect 1, wherein the variable domain is a human     variable domain.     -   A human variable domain is, for example, the product of         recombination in a transgenic non-human vertebrate of human VH,         D and JH gene segments. Alternatively, the variable domain is         identified using in vitro display technology from a human VH         library, e.g., using phage display, ribosome display or yeast         display, as is known in the art.     -   In another embodiment, the variable domain is a humanized         variable domain, e.g., comprising human frameworks with         non-human (e.g., mouse or rat) CDRs). Humanization technology is         conventional in the art, and will be readily known to the         skilled person.

-   3. The method of any preceding aspect, wherein the variable domain     has previously been selected from a non-human vertebrate that has     been immunised with the antigen.     -   For example, the vertebrate (such as a mouse or rat) genome         comprises a chimaeric heavy chain locus comprising a human         variable region (human V, D and JH gene segments) operably         connected upstream of a non-human vertebrate constant region so         that the locus is able to rearrange for the expression of heavy         chains comprising human variable domains and non-human         vertebrate constant regions.     -   In alternative embodiments, the variable domain is selected         using an in vitro technology such as phage display, ribosome         display or yeast display. In this case the variable domain may         be displayed with or without an constant region, provided that         it is later combined with a human constant region as per the         invention.

-   4. The method of any preceding aspect, comprising providing an     expression vector (E.g., a mammalian expression vector, such as a     CHO or HEK293 vector) comprising a nucleotide sequence encoding the     constant region; inserting a nucleotide sequence encoding the     variable domain into the vector 5′ of the constant region sequence;     inserting the vector into a host cell and expressing the heavy chain     by the host cell; the method further comprising isolating a heavy     chain (e.g., as part of an antibody) comprising the variable domain     and the human constant region.     -   The vector comprises regulatory elements sufficient to effect         expression of the heavy chain when the vector is harboured by a         host cell, e.g., a CHO or HEK293 cell.

-   5. The method of any preceding aspect, further comprising obtaining     a nucleotide sequence encoding the heavy chain.

-   6. An antibody comprising a human heavy chain, the heavy chain     comprising a variable domain that is specific for an antigen and a     constant region that is an IGHAref, IGHA1a, IGHA2a, IGHA2b,     IGHG1ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref,     IGHG4a, IGHDref, IGHEref, IGHMref, IGHMa or IGHMb constant region.

-   7. A polypeptide comprising (in N- to C-terminal direction) a leader     sequence, a human variable domain that is specific for an antigen     and a human constant region that is an IGHAref, IGHA1a, IGHA2a,     IGHA2b, IGHG1ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b,     IGHG4ref, IGHG4a, IGHDref, IGHEref, IGHMref, IGHMa or IGHMb constant     region; wherein (i) the leader sequence is not the native human     variable domain leader sequence (e.g., the leader sequence is     another human leader sequence or a non-human leader sequence);     and/or (ii) the variable domain comprises mouse AID-pattern somatic     mutations or mouse terminal deoxynucleotidyl transferase     (TdT)-pattern junctional mutations.

-   8. A nucleotide sequence encoding (in 5′ to 3′ direction) a leader     sequence and a human antibody heavy chain, the heavy chain     comprising a variable domain that is specific for an antigen and a     constant region that is an IGHAref, IGHA1a, IGHA2a, IGHA2b,     IGHG1ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref,     IGHG4a, IGHDref, IGHEref, IGHMref, IGHMa or IGHMb constant region;     and the leader sequence being operable for expression (e.g., in a     mammalian CHO or HEK293 cell) of the heavy chain and wherein the     leader sequence is not the native human variable domain leader     sequence (e.g., the leader sequence is another human leader sequence     or a non-human leader sequence).     -   In an example, the leader sequence is

ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCCACCGCCACCGGCG TGCACAGC

-   -   Which translates to

MGWSCIILFLVATATGVHS

-   9. A nucleotide sequence encoding (in 5′ to 3′ direction) a promoter     and a human antibody heavy chain, the heavy chain comprising a     variable domain that is specific for an antigen and a constant     region that is an IGHAref, IGHA1a, IGHA2a, IGHA2b, IGHG1ref,     IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref, IGHG4a,     IGHDref, IGHEref, IGHMref, IGHMa or IGHMb constant region; and the     promoter being operable for expression (e.g., in a mammalian CHO or     HEK293 cell) of the heavy chain and wherein the promoter is not the     native human promoter.     -   In one embodiment, the promoter sequence is a human IGK 3-15         promoter. -   10. The antibody, polypeptide or nucleotide sequence of any one of     aspects 6 to 9, wherein the variable domain comprises mouse     AID-pattern somatic mutations and/or mouse terminal deoxynucleotidyl     transferase (TdT)-pattern junctional mutations. -   For example, one way, in any aspect of this configuration of the     invention, to provide mouse AID-pattern somatic mutations and/or     mouse terminal deoxynucleotidyl transferase (TdT)-pattern junctional     mutations is to select a variable domain from a non-human vertebrate     or cell. For example, a vertebrate or cell as disclosed herein. -   11. A vector (e.g., a CHO cell or HEK293 cell vector) comprising the     nucleic acid of aspect 8, 9 or 10; optionally wherein the vector is     in a host cell (e.g., a CHO cell or HEK293 cell). -   12. A pharmaceutical composition comprising the antibody or     polypeptide of any one of aspects 6, 7 and 10, together with a     pharmaceutically-acceptable excipient, diluent or a medicament     (e.g., a further antigen-specific variable domain, antibody chain or     antibody). -   13. The antibody or polypeptide of any one of aspects 6, 7 and 10     for use in treating and/or preventing a medical condition in a human     patient. -   14. Use of the antibody or polypeptide of any one of aspects 6, 7     and 10 for the manufacture of a medicament for treating and/or     preventing a medical condition in a human patient. -   15. The antibody, polypeptide or use of aspect 13 or 14, wherein the     human is a member of a human population selected from population     numbers 1-14, wherein the populations are numbered as follows     (population labels being according to 1000 Genomes Project     nomenclature)

1=ASW; 2=CEU; 3=CHB; 4=CHS; 5=CLM; 6=FIN; 7=GBR; 8=IBS; 9=JPT; 10=LWK; 11=MXL; 12=PUR; 13=TSI; 14=YRI.

-   16. The antibody, polypeptide or use of aspect 15, wherein the     constant region is a     -   (i) IGHA1a constant region and the human population is selected         from any population number 1-14;     -   (ii) IGHA2a constant region and the human population is selected         from any population number 1-14;     -   (iii) IGHA2b constant region and the human population is         selected from any population number 1-14;     -   (iv) IGHG2a constant region and the human population is selected         from any population number 1-9 and 11-13;     -   (v) IGHG3a constant region and the human population is selected         from any population number 1-14;     -   (vi) IGHG3b constant region and the human population is selected         from any population number 1-8 and 11-13;     -   (vii) IGHG4a constant region and the human population is         selected from any population number 1-9 and 11-13;     -   (viii) IGHMa constant region and the human population is         selected from any population number 1-14; or     -   (ix) IGHMb constant region and the human population is selected         from any population number 1-14;

Wherein the populations are numbered as follows (population labels being according to 1000 Genomes Project nomenclature)

1=ASW; 2=CEU; 3=CHB; 4=CHS; 5=CLM; 6=FIN; 7=GBR; 8=IBS; 9=JPT; 10=LWK; 11=MXL; 12=PUR; 13=TSI; 14=YRI.

-   17. A vector (e.g., a CHO cell or HEK293 cell vector) comprising a     IGHG1ref, IGHG2ref, IGHG2a, IGHG3ref, IGHG3a, IGHG3b, IGHG4ref or     IGHG4a constant region nucleotide sequence that is 3′ of a cloning     site for the insertion of a human antibody heavy chain variable     domain nucleotide sequence, such that upon insertion of such a     variable domain sequence the vector comprises (in 5′ to 3′     direction) a promoter, a leader sequence, the variable domain     sequence and the constant region sequence so that the vector is     capable of expressing a human antibody heavy chain when present in a     host cell.

The Present Invention Provides in a Sixth Configuration— Multiple Variants in the Same Genome C is or Trans

The inventors' analysis has revealed groupings of naturally-occurring human antibody gene segment variants as set out in Table 13 and Table 14. This revealed the possibility of producing transgenic genomes in non-human vertebrates and cells wherein the genomes contain more than the natural human complement of specific human gene segments. In one example, this can be achieved by providing more than the natural human complement of a specific gene segment type on one or both of the respective Ig locus (e.g., one or both chromosomes harbouring IgH in a mouse genome or mouse cell genome).

To this end, this configuration of the invention provides the following (as set out in numbered paragraphs):—

-   1. A non-human vertebrate (e.g., a mouse or rat) or a non-human     vertebrate cell (e.g., an ES cell or a B-cell) having a genome     comprising at least 3 human variable region gene segments of the     same type (e.g., at least 3 human VH6-1 gene segments, at least 3     human JH6 gene segments, at least 3 human VK1-39 gene segments, at     least 3 human D2-2 gene segments or at least 3 human JK1 gene     segments), wherein at least two of the human gene segments are     variants that are not identical to each other.     -   For example, the genome comprises a variable region that         comprises V, D and J gene segments (for the variable region of a         heavy chain locus) or V and J gene segments (for the variable         region of a light chain locus) upstream of a constant region for         expression of heavy or light chains respectively.     -   In an alternative, the skilled person can choose to provide more         than the wild type human complement of a specific gene segment         type by providing several copies of one variant type of the         human gene segment. Thus, there is provided A non-human         vertebrate (e.g., a mouse or rat) or a non-human vertebrate cell         (e.g., an ES cell or a B-cell) having a genome comprising at         least 3 human variable region gene segments of the same type         (e.g., at least 3 human VH6-1 gene segments, at least 3 human         JH6 gene segments, at least 3 human VK1-39 gene segments, at         least 3 human D2-2 gene segments or at least 3 human JK1 gene         segments), wherein the human gene segments are identical         variants.     -   For example, the genome comprises a variable region that         comprises V, D and J gene segments (for the variable region of a         heavy chain locus) or V and J gene segments (for the variable         region of a light chain locus) upstream of a constant region for         expression of heavy or light chains respectively. -   2. A non-human vertebrate (e.g., a mouse or rat) or a non-human     vertebrate cell (e.g., an ES cell or a B-cell) having a genome     comprising at least 2 different non-endogenous variable region gene     segments of the same type (e.g., at least 2 human VH6-1 gene     segments, at least 3 human JH6 gene segments, at least 2 human     VK1-39 gene segments, at least 2 human D2-2 gene segments or at     least 2 human JK1 gene segments) cis at the same Ig locus.     -   In an alternative, the skilled person can choose to provide more         than the wild type human complement of a specific gene segment         type by providing several copies of one variant type of the         human gene segment. Thus, there is provided     -   A non-human vertebrate (e.g., a mouse or rat) or a non-human         vertebrate cell (e.g., an ES cell or a B-cell) having a genome         comprising at least 2 non-endogenous variable region gene         segments of the same variant type (e.g., at least 2 human JH6*02         gene segments) cis at the same Ig locus. -   3. A non-human vertebrate (e.g., a mouse or rat) or a non-human     vertebrate cell (e.g., an ES cell or a B-cell) having a genome     comprising at least 2 different human variable region gene segments     of the same type (e.g., at least 2 human VH6-1 gene segments, at     least 2 human JH6 gene segments, at least 2 human VK1-39 gene     segments, at least 2 human D2-2 gene segments or at least 2 human     JK1 gene segments) trans at the same Ig locus; and optionally a     third human gene segment of the same type, wherein the third gene     segment is cis with one of said 2 different gene segments.     -   In an alternative, the skilled person can choose to provide more         than the wild type human complement of a specific gene segment         type by providing several copies of one variant type of the         human gene segment. Thus, there is provided A non-human         vertebrate (e.g., a mouse or rat) or a non-human vertebrate cell         (e.g., an ES cell or a B-cell) having a genome comprising at         least 2 different human variable region gene segments of the         same variant type (e.g., at least 2 human JH6*02 gene segments)         trans at the same Ig locus; and optionally a third human gene         segment of the same variant type, wherein the third gene segment         is cis with one of said 2 different gene segments. -   4. A population of non-human vertebrates (e.g., mice or rats)     comprising a repertoire of human variable region gene segments,     wherein the plurality comprises at least 2 human variable region     gene segments of the same type (e.g., at least 2 human VH6-1 gene     segments, at least 2 human JH6 gene segments, at least 2 human     VK1-39 gene segments, at least 2 human D2-2 gene segments or at     least 2 human JK1 gene segments), a first of said different gene     segments is provided in the genome of a first vertebrate of the     population, and a second of said different gene segments being     provided in the genome of a second vertebrate of the population,     wherein the genome of the first vertebrate does not comprise the     second gene segment. -   5. A non-human vertebrate (e.g., a mouse or rat) or a non-human     vertebrate cell (e.g., an ES cell or a B-cell) having a genome     comprising at least 2 different non-endogenous variable region gene     segments of the same type (e.g., at least 2 human VH6-1 gene     segments, at least 2 human JH6 gene segments, at least 2 human     VK1-39 gene segments, at least 2 human D2-2 gene segments or at     least 2 human JK1 gene segments), wherein the gene segments are     derived from the genome sequence of different human individuals that     are not genetically related over at least 3 generations. -   6. A method of enhancing the human immunoglobulin gene diversity of     a non-human vertebrate (e.g., a mouse or rat), the method comprising     providing the vertebrate with a genome comprising at least 3 human     variable region gene segments of the same type (e.g., at least 3     human VH6-1 gene segments, at least 3 human JH6 gene segments, at     least 3 human VK1-39 gene segments, at least 3 human D2-2 gene     segments or at least 3 human JK1 gene segments), wherein at least     two of the human gene segments are variants that are not identical     to each other. -   7. A method of enhancing the immunoglobulin gene diversity of a     non-human vertebrate (e.g., a mouse or rat), the method comprising     providing the vertebrate with a genome comprising at least 2     different non-endogenous variable region gene segments of the same     type (e.g., at least 2 human VH6-1 gene segments, at least 2 human     JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2     human D2-2 gene segments or at least 2 human JK1 gene segments) cis     at the same Ig locus. -   8. A method of enhancing the immunoglobulin gene diversity of a     non-human vertebrate (e.g., a mouse or rat), the method comprising     providing the vertebrate with a genome comprising at least 2     different human variable region gene segments of the same type     (e.g., at least 2 human VH6-1 gene segments, at least 2 human JH6     gene segments, at least 2 human VK1-39 gene segments, at least 2     human D2-2 gene segments or at least 2 human JK1 gene segments)     trans at the same Ig locus; and optionally a third human gene     segment of the same type, wherein the third gene segment is cis with     one of said 2 different gene segments. -   9. A method of providing an enhanced human immunoglobulin variable     region gene segment repertoire, the method comprising providing a     population of non-human vertebrates (e.g., a mouse or rat)     comprising a repertoire of human variable region gene segments,     wherein the method comprises providing at least 2 different human     variable region gene segments of the same type (e.g., at least 2     human VH6-1 gene segments, at least 2 human JH6 gene segments, at     least 2 human VK1-39 gene segments, at least 2 human D2-2 gene     segments or at least 2 human JK1 gene segments), wherein a first of     said different gene segments is provided in the genome of a first     vertebrate of the population, and a second of said different gene     segments is provided in the genome of a second vertebrate of the     population, wherein the genome of the first vertebrate does not     comprise the second gene segment. -   10. A method of enhancing the human immunoglobulin gene diversity of     a non-human vertebrate (e.g., a mouse or rat), the method comprising     providing the vertebrate with a genome comprising at least 2     different non-endogenous variable region gene segments of the same     type (e.g., at least 2 human VH6-1 gene segments, at least 2 human     JH6 gene segments, at least 2 human VK1-39 gene segments, at least 2     human D2-2 gene segments or at least 2 human JK1 gene segments),     wherein the gene segments are derived from the genome sequence of     different human individuals that are not genetically related over at     least 3 generations. -   11. The vertebrate, cell or method of any preceding paragraph,     wherein at least 2 or 3 of said different gene segments are provided     cis at the same Ig locus in said genome. -   12. The vertebrate, cell or method of any preceding paragraph,     wherein the gene segments are derived from the genome sequence of     different human individuals that are not genetically related over at     least 3 generations. -   13. The vertebrate, cell or method of any preceding paragraph,     wherein the gene segments are derived from the genome sequence of     two or more different human individuals; optionally wherein the     different human individuals are from different human populations. -   14. The vertebrate, cell or method of paragraph 13, wherein the     individuals are not genetically related. -   15. A method of enhancing the human immunoglobulin gene diversity of     a non-human vertebrate (e.g., a mouse or rat), the method comprising     providing the vertebrate with a genome comprising at least 2 human     variable region gene segments of the same type (e.g., at least 2     human VH6-1 gene segments, at least 2 human JH6 gene segments, at     least 2 human VK1-39 gene segments, at least 2 human D2-2 gene     segments or at least 2 human JK1 gene segments), wherein the gene     segments are derived from the genome sequence of different human     individuals that are not genetically related over at least 3     generations; optionally wherein at least 2 or 3 of said different     gene segments are provided at the same Ig locus in said genome. -   16. The method of paragraph 15, wherein the different human     individuals are from different human populations. -   17. The method of paragraph 15, wherein the individuals are not     genetically related. -   18. The vertebrate, cell or method of preceding paragraph, wherein     at least one of the different segments is a synthetic mutant of a     human germline gene segment. -   19. The vertebrate, cell or method of any preceding paragraph,     wherein each of said gene segments occurs in 10 or more different     human populations. -   20. The vertebrate, cell or method of preceding paragraph, wherein     each of said gene segments has a human frequency of 5% or greater     (e.g., 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,     90, or 95% or greater).     -   In this respect, the skilled person can be guided by the         information provided in Table 14. Frequency can, for example, be         cumulative frequency in the 1000 Genomes database. -   21. The vertebrate, cell or method of paragraph 20, wherein each of     said gene segments occurs in 10 or more different human populations. -   22. The vertebrate, cell or method of any preceding paragraph,     wherein each of said gene segments occurs in the 1000 Genomes     database in more than 50 individuals. -   23. The vertebrate, cell or method of preceding paragraph, wherein     each of said gene segments (i) has a human frequency of 5% or     greater (e.g., 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,     80, 85, 90, or 95% or greater); and (ii) occurs in 10 or more     different human populations.     -   In this respect, the skilled person can be guided by the         information provided in Table 14.     -   Frequency can, for example, be cumulative frequency in the 1000         Genomes database. -   24. A non-human vertebrate (e.g., a mouse or rat) or a non-human     vertebrate cell (e.g., an ES cell or a B-cell) having a genome     comprising first and second human Ig locus gene segments of the same     type (e.g., first and second human JH6 gene segments; or first and     second IgG2 gene segments; or first and second human JA7 gene     segments), wherein the first gene segment is a gene segment selected     from Table 14 (e.g., IGHJ6-a) and the second gene segment is the     corresponding reference sequence (e.g., IGHJ6 ref; SEQ ID NO: 244).     -   Table 14 lists commonly-occurring natural human variants. It can         be seen that these occur across many human populations and thus         usefully have wide applicability for human antibody-based drugs.     -   For example, the gene segments are provided as targeted         insertions into an endogenous non-human vertebrate Ig locus.         Alternatively, random integration (e.g., using YACs) as is know         in the art can be performed.     -   For example, the genome comprises a variable region that         comprises V, D and J gene segments (for the variable region of a         heavy chain locus) or V and J gene segments (for the variable         region of a light chain locus) upstream of a constant region for         expression of heavy or light chains respectively.     -   In another embodiment, the invention enables the skilled person         to select two or more different naturally-occurring human gene         segment variants for combination into the genome of a non-human         vertebrate or cell. A reference sequence need not be included.         It may be desirable to use one or more rare gene segments to         increase diversity of the repertoire. Additionally or         alternatively, it may be desirable to include a mixture of         frequent and rare variants of the same type to provide         repertoire diversity. The variants may be chosen additionally or         alternatively to tailor the gene segment inclusion to one or         more specific human populations as indicated by the information         provided in Table 13 or Table 14.     -   Thus, the invention provides     -   A non-human vertebrate (e.g., a mouse or rat) or a non-human         vertebrate cell (e.g., an ES cell or a B-cell) having a genome         comprising first and second human Ig locus gene segments of the         same type (e.g., first and second human JH6 gene segments; or         first and second IgG2 gene segments; or first and second human         JA7 gene segments), wherein the gene segments are gene segments         selected from Table 13 or Table 14; and         optionally wherein one or more of the gene segments appears in         Table 14 (e.g., IGHJ6-a) or is a reference sequence (e.g., IGHJ6         ref; SEQ ID NO: 244). -   25. The vertebrate or cell of paragraph 24, wherein the genome     comprises a third human gene segment of said type, the third gene     segment being different from the first and second gene segments. -   26. The vertebrate or cell of paragraph 24 or 25, wherein the first     and second gene segments are cis on the same chromosome; and     optionally the third gene segment is also cis on said chromosome. -   27. The vertebrate or cell of paragraph 26, wherein the gene     segments are targeted insertions into an endogenous non-human Ig     locus.     -   For example, the gene segments are heavy chain gene segments and         the non-human locus is an IgH locus. For example, the gene         segments are light chain (kappa or lambda) gene segments and the         non-human locus is an IgL locus. -   28. The vertebrate or cell of paragraph 24 or 25, wherein the first     and second gene segments are trans on different chromosomes.     -   Thus, the chromosomes are the same type (e.g., both mouse         chromosome 6 or rat chromosome 4). -   29. The vertebrate or cell of any one of paragraphs 24 to 28,     wherein the first gene segment is a gene segment selected from any     one of Tables 1 to 7 and 9 to 14 (e.g., selected from Table 13     or 14) and the second gene segment is the corresponding reference     sequence. -   30. A population of non-human vertebrates (e.g., mice or rats)     comprising first and second human Ig locus gene segments of the same     type (e.g., first and second human JH6 gene segments; or first and     second IgG2 gene segments; or first and second human JA7 gene     segments), wherein the first gene segment is a gene segment selected     from any one of Tables 1 to 7 and 9 to 14 (e.g., Table 13 or 14)     (e.g., IGHJ6-a) and the second gene segment is the corresponding     reference sequence (e.g., SEQ ID NO: 7), wherein the first gene     segment is provided in the genome of a first vertebrate of the     population, and the second gene segment is provided in the genome of     a second vertebrate of the population. -   31. The population of paragraph 30, wherein the genome of the first     vertebrate does not comprise the second gene segment. -   32. The population of paragraph 30 or 31, wherein the population     comprises a third human gene segment of said type, the third gene     segment being different from the first and second gene segments and     optionally wherein the first and third gene segments are present in     the genome of the first vertebrate. -   33. The population of paragraph 30, 31 or 32, wherein the gene     segments are targeted insertions into an endogenous non-human Ig     locus in the respective genome.     -   For example, the gene segments are heavy chain gene segments and         the non-human locus is an IgH locus. For example, the gene         segments are light chain (kappa or lambda) gene segments and the         non-human locus is an IgL locus. -   34. The population of any one of paragraphs 30 to 33, wherein the     first gene segment is a gene segment selected from any one of Tables     1 to 7 and 9 to 14 (e.g., Table 13 or 14) and the second gene     segment is the corresponding reference sequence. -   35. A method of enhancing the human immunoglobulin gene diversity of     a non-human vertebrate (e.g., a mouse or rat), the method comprising     providing the vertebrate with a genome comprising first and second     human Ig locus gene segments of the same type (e.g., first and     second human JH6 gene segments; or first and second IgG2 gene     segments; or first and second human JA7 gene segments), wherein the     first gene segment is a gene segment selected from any one of Tables     1 to 7 and 9 to 14 (e.g., Table 13 or 14) (e.g., IGHJ6-a) and the     second gene segment is the corresponding reference sequence (e.g.,     SEQ ID NO: 7). -   36. A method of providing an enhanced human immunogolobulin gene     segment repertoire, the method comprising providing a population     according to any one of paragraphs 30 to 33.

Variants Prevalent in Few Populations

In another aspect, it is of note that certain human gene segment variants may appear relatively frequently in one or a small number of populations, but is not found prevalently across many different human populations. There is thinking that specific germline gene segment repertoires have evolved in individual human ethnic populations due to iterative exposure to antigens (e.g., disease pathogen antigens) to which the population is often exposed. Repeated exposure and mutation may have lead to the evolution of gene segment variants that can provide an effective response to the antigen (pathogen) in the population, and this may explain the conservation of the gene segments in those populations (as opposed to other human ethnic populations that may not have frequently encountered the antigen). With this in mind, the inventors identified gene segment variants from their analysis that are relatively prevalent in a small number of human populations, and not across many populations. The inventors realized that inclusion of one or more of such gene segments in the configurations of the invention (e.g., in transgenic Ig loci, vertebrates and cells) would be useful for producing antibodies, Ig chains and variable domains that can address antigens (e.g., disease-causing antigens or pathogens) to which the small number of human populations may become exposed. Such products would be useful for treating and/or preventing disease or medical conditions in members of such a population. This aspect could also be useful for addressing infectious disease pathogens that may have been common in the small number of populations, but which in the future or relatively recently in evolution has become a more prevalent disease-causing pathogen in other human populations (i.e., those not listed in Table 13 against the gene segment variant(s) in question). To this end, from the 1000 Genomes database the inventors have identified the gene segment variants listed in Table 20.

Thus, according to any configuration or aspect described herein, one, more or all of the gene segments used in the present invention can be a gene segment listed in Table 20A, 20B, 20C or 20D.

Multiple JH Gene Segment Variants

A specific application of this configuration is the provision of multiple human JH gene segments as follows.

A non-human vertebrate (e.g., a mouse or rat) or a non-human vertebrate cell (e.g., an ES cell or a B-cell) having a genome comprising at least 3 human JH gene segments of the same type (JH1, JH2, JH3, JH4, JH5 or JH6), wherein at least two of the human JH gene segments are variants that are not identical to each other.

In an example, any cell of the invention is an isolated cell. An “isolated” cell is one that has been identified, separated and/or recovered from a component of its production environment (e.g., naturally or recombinantly). Preferably, the isolated cell is free of association with all other components from its production environment, e.g., so that the cell can produce an antibody to an FDA-approvable or approved standard. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the resultant antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Ordinarily, however, an isolated cell will be prepared by at least one purification step.

A non-human vertebrate (e.g., a mouse or rat) or a non-human vertebrate cell (e.g., an ES cell or a B-cell) having a genome comprising at least 2 different non-endogenous JH gene segments (e.g., human gene segments) of the same type (JH1, JH2, JH3, JH4, JH5 or JH6) cis at the same Ig (e.g., IgH, e.g., endogenous IgH, e.g., mouse or rat IgH) locus. In an example, the genome comprises a human VH, D and JH repertoire comprising said different JH gene segments. Optionally the non-endogenous JH gene segments are non-mouse or non-rat, e.g., human JH gene segments. In an example one or more or all of the non-endogenous gene segments are synthetic.

A non-human vertebrate (e.g., a mouse or rat) or a non-human vertebrate cell (e.g., an ES cell or a B-cell) having a genome comprising at least 2 different human JH gene segments of the same type (JH1, JH2, JH3, JH4, JH5 or JH6) trans at the same Ig (e.g., IgH, e.g., endogenous IgH, e.g., mouse or rat IgH) locus; and optionally a third human JH gene segments of the same type, wherein the third JH is cis with one of said 2 different JH gene segments.

A population of non-human vertebrates (e.g., mice or rats) comprising a repertoire of human JH gene segments, wherein the plurality comprises at least 2 different human JH gene segments of the same type (JH1, JH2, JH3, JH4, JH5 or JH6), a first of said different JH gene segments is provided in the genome of a first vertebrate of the population, and a second of said different JH gene segments being provided in the genome of a second vertebrate of the population, wherein the genome of the first vertebrate does not comprise the second JH gene segment.

A non-human vertebrate (e.g., a mouse or rat) or a non-human vertebrate cell (e.g., an ES cell or a B-cell) having a genome comprising at least 2 different non-endogenous (e.g., human) JH gene segments of the same type (JH1, JH2, JH3, JH4, JH5 or JH6), wherein the JH gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations (e.g., 3, 4, 5 or 6 generations). Optionally the non-endogenous JH gene segments are human JH gene segments. In an example one or more or all of the non-endogenous gene segments are synthetic.

A method of enhancing the human immunoglobulin gene diversity of a non-human vertebrate (e.g., a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 3 human JH gene segments of the same type (JH1, JH2, JH3, JH4, JH5 or JH6), wherein at least two of the human JH gene segments are variants that are not identical to each other.

A method of enhancing the immunoglobulin gene diversity of a non-human vertebrate (e.g., a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 different non-endogenous (e.g., human) JH gene segments of the same type (JH1, JH2, JH3, JH4, JH5 or JH6) cis at the same Ig (e.g., IgH, e.g., endogenous IgH, e.g., mouse or rat IgH) locus). Optionally the non-endogenous JH gene segments are non-mouse or non-rat, e.g., human JH gene segments. In an example one or more or all of the non-endogenous gene segments are synthetic.

A method of enhancing the immunoglobulin gene diversity of a non-human vertebrate (e.g., a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 different human JH gene segments of the same type (JH1, JH2, JH3, JH4, JH5 or JH6) trans at the same Ig (e.g., IgH, e.g., endogenous IgH, e.g., mouse or rat IgH) locus; and optionally a third human JH gene segments of the same type, wherein the third JH is cis with one of said 2 different JH gene segments.

A method of providing an enhanced human immunoglobulin JH gene segment repertoire, the method comprising providing a population of non-human vertebrates (e.g., a mouse or rat) comprising a repertoire of human JH gene segments, wherein the method comprises providing at least 2 different human JH gene segments of the same type (JH1, JH2, JH3, JH4, JH5 or JH6), wherein a first of said different JH gene segments is provided in the genome of a first vertebrate of the population, and a second of said different JH gene segments is provided in the genome of a second vertebrate of the population, wherein the genome of the first vertebrate does not comprise the second JH gene segment.

A method of enhancing the human immunoglobulin gene diversity of a non-human vertebrate (e.g., a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 different non-endogenous (e.g., human) JH gene segments of the same type (JH1, JH2, JH3, JH4, JH5 or JH6), wherein the JH gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations (e.g., 3, 4, 5, or 6 generations). Optionally the non-endogenous JH gene segments are human JH gene segments. In an example one or more or all of the non-endogenous gene segments are synthetic.

In an example of the vertebrate or cell or the method of the invention at least 2 or 3 of said different gene segments are provided cis at the same Ig locus in said genome.

In an example of the vertebrate or cell or the method of the invention the JH gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations (e.g., 3, 4, 5, or 6 generations).

In an example of the vertebrate or cell or the method of the invention the JH gene segments are derived from the genome sequence of two or more different human individuals; optionally wherein the different human individuals are from different human populations.

In an example of the vertebrate or cell or the method of the invention the individuals are not genetically related (e.g., going back 3, 4, 5, or 6 generations).

In an example of the vertebrate or cell or the method of the invention at least one of the different JH segments is a synthetic mutant of a human germline JH gene segment.

The invention also provides a method of enhancing the human immunoglobulin gene diversity of a non-human vertebrate (e.g., a mouse or rat), the method comprising providing the vertebrate with a genome comprising at least 2 human JH gene segments of the same type (JH1, JH2, JH3, JH4, JH5 or JH6), wherein the JH gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations (e.g., 3, 4, 5, or 6 generations); optionally wherein at least 2 or 3 of said different gene segments are provided at the same IgH locus in said genome.

In an example of the vertebrate or cell or the method of this embodiment of the invention the genome comprises a substantially complete functional repertoire of human JH gene segment types supplemented with one, two or more human JH gene segments, wherein said substantially complete functional repertoire and the supplementary JH gene segments are not found together in the germline genome of a human individual.

In an example of the population of the invention, the population comprises a substantially complete functional repertoire of human JH gene segment types supplemented with one, two or more human JH gene segments, wherein said substantially complete functional repertoire and the supplementary JH gene segments are not found together in the germline genome of a human individual.

A non-human vertebrate (e.g., a mouse or rat) or a non-human cell (e.g., an ES cell or a B-cell) having a genome comprising a substantially complete functional repertoire of human JH gene segment types supplemented with one, two or more human JH gene segments, wherein said substantially complete functional repertoire and the supplementary JH gene segments are not found together in the germline genome of a human individual.

A population of non-human vertebrates (e.g., mice or rats) comprising a substantially complete functional repertoire of human JH gene segment types supplemented with one, two or more human JH gene segments, wherein said substantially complete functional repertoire and the supplementary JH gene segments are not found together in the germline genome of a human individual.

In an example of the vertebrate or the population, at least one of said JH gene segments is SEQ ID NO: 1, 2, 3 or 4. For example, at least one of said JH gene segments is SEQ ID NO: 1 and at least one, two or more of said supplementary JH gene segments is a variant according to any example above. For example, at least one of said JH gene segments is SEQ ID NO: 2 and at least one, two or more of said supplementary JH gene segments is a variant according to any one of the examples above. For example, at least one of said JH gene segments is SEQ ID NO: 2 and at least one, two or more of said supplementary JH gene segments is a variant according to any one of the examples above.

In an embodiment, the non-human vertebrate or vertebrate cell of the invention comprises a genome that comprises VH, D and JH gene repertoires comprising human gene segments, the JH gene repertoire (e.g., a human JH gene segment repertoire) comprising a plurality of JH1 gene segments provided by at least 2 different JH1 gene segments in cis at the same Ig locus in said genome;

a plurality of JH2 gene segments provided by at least 2 different JH2 gene segments in cis at the same Ig locus in said genome; a plurality of JH3 gene segments provided by at least 2 different JH3 gene segments in cis at the same Ig locus in said genome; a plurality of JH4 gene segments provided by at least 2 different JH4 gene segments in cis at the same Ig locus in said genome; a plurality of JH5 gene segments provided by at least 2 different JH5 gene segments in cis at the same Ig locus in said genome; and/or a plurality of JH6 gene segments provided by at least 2 different JH6 gene segments in cis at the same Ig locus in said genome; optionally wherein the JH gene segments are derived from the genome sequence of two or more different human individuals.

Optionally said at least 2 different JH gene segments are human gene segments or synthetic gene segments derived from human gene segments.

Optionally, the Ig locus is a IgH locus, e.g., an endogenous locus, e.g., a mouse or rat IgH locus.

In an embodiment, the non-human vertebrate or vertebrate cell of the invention comprises a genome that comprises VH, D and JH gene repertoires comprising human gene segments, the JH gene repertoire (e.g., a human JH gene segment repertoire) comprising a plurality of JH1 gene segments provided by at least 3 different JH1 gene segments; a plurality of JH2 gene segments provided by at least 3 different JH2 gene segments; a plurality of JH3 gene segments provided by at least 3 different JH3 gene segments; a plurality of JH4 gene segments provided by at least 3 different JH4 gene segments; a plurality of JH5 gene segments provided by at least 3 different JH5 gene segments; and/or a plurality of JH6 gene segments provided by at least 3 different JH6 gene segments; optionally wherein the JH gene segments are derived from the genome sequence of two or three different human individuals;

optionally wherein at least 2 or 3 of said different gene segments are provided in cis at the same Ig locus in said genome.

Optionally said at least 3 different JH gene segments are human gene segments or synthetic gene segments derived from human gene segments.

Optionally, the Ig locus is a IgH locus, e.g., an endogenous locus, e.g., a mouse or rat IgH locus.

Optionally in the vertebrate or cell the different human individuals are from different human populations.

Optionally in the vertebrate or cell the individuals are not genetically related (e.g., Going back 3, 4, 5 or 6 generations).

Optionally in the vertebrate or cell at least one of the different JH segments is a synthetic mutant of a human germline JH gene segment.

In an embodiment of a non-human vertebrate or vertebrate cell (optionally an ES cell or B-cell) according to the invention, the vertebrate or cell genome comprises human VH, D and JH gene repertoires, the JH gene repertoire (e.g., a human JH gene repertoire) comprising a plurality of JH1 gene segments provided by at least 2 different human JH1 gene segments, optionally in cis at the same Ig locus in said genome;

a plurality of JH2 gene segments provided by at least 2 different human JH2 gene segments, optionally in cis at the same Ig locus in said genome; a plurality of JH3 gene segments provided by at least 2 different human JH3 gene segments, optionally in cis at the same Ig locus in said genome; a plurality of JH4 gene segments provided by at least 2 different human JH4 gene segments, optionally in cis at the same Ig locus in said genome; a plurality of JH5 gene segments provided by at least 2 different human JH5 gene segments, optionally in cis at the same Ig locus in said genome; and/or a plurality of JH6 gene segments provided by at least 2 different human JH6 gene segments, optionally in cis at the same Ig locus in said genome; wherein the JH gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations (e.g., 3, 4, 5 or 6 generations).

Optionally said at least 2 different JH gene segments are human gene segments or synthetic gene segments derived from human gene segments.

Optionally, the Ig locus is a IgH locus, e.g., an endogenous locus, e.g., a mouse or rat IgH locus. Optionally in the vertebrate or cell the human individuals are from different human populations.

JH5

An embodiment provides a vertebrate, cell or population of the invention whose genome comprises a plurality of JH5 gene segments, wherein the plurality comprises a human JH5 gene variant of SEQ ID NO: 1, wherein the variant comprises a nucleotide mutation at one or more positions corresponding to positions

106,330,024 106,330,027 106,330,032 106,330,041 106.330.44 106.330.45 106.330.62 106.330.63 106.330.65 106.330.66 106.330.67 106.330.68 and 106,330,071 on human chromosome 14.

In the vertebrate, cell or population optionally the plurality comprises a human JH5 gene variant of SEQ ID NO: 1, wherein the variant comprises a guanine at a position corresponding to position 106,330,067 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the variant comprises additionally a mutation at a position corresponding to (i) position 106,330,071 on human chromosome 14 (optionally the additional mutation being a guanine); (ii) position 106,330,066 on human chromosome 14 (optionally the additional mutation being a guanine); and/or (iii) position 106,330,068 on human chromosome 14 (optionally the additional mutation being a thymine).

Optionally the plurality comprises a human JH5 gene variant of SEQ ID NO: 1, wherein the variant comprises a guanine at a position corresponding to position 106,330,071 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the variant comprises additionally a mutation at a position corresponding to (i) position 106,330,063 on human chromosome 14 (optionally the additional mutation being an adenine); and/or (ii) position 106,330,067 on human chromosome 14 (optionally the additional mutation being a guanine).

Optionally the plurality comprises a human JH5 gene variant of SEQ ID NO: 1, wherein the variant comprises a cytosine at a position corresponding to position 106,330,045 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the plurality comprises a human JH5 gene variant of SEQ ID NO: 1, wherein the variant comprises an adenine at a position corresponding to position 106,330,044 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the variant comprises additionally a mutation at a position corresponding to (i) position 106.330.66 on human chromosome 14 (optionally the additional mutation being a guanine); and/or (ii) position 106,330,068 on human chromosome 14 (optionally the additional mutation being a thymine).

Optionally the plurality comprises a human JH5 gene variant of SEQ ID NO: 1, wherein the variant comprises a guanine at a position corresponding to position 106,330,066 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the variant comprises additionally a mutation at a position corresponding to (i) position 106.330.67 on human chromosome 14 (optionally the additional mutation being a guanine); and/or (ii) position 106,330,068 on human chromosome 14 (optionally the additional mutation being a thymine).

Optionally the plurality comprises a human JH5 gene variant of SEQ ID NO: 1, wherein the variant comprises a thymine at a position corresponding to position 106,330,068 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the variant comprises additionally a mutation at a position corresponding to (i) position 106,330,067 on human chromosome 14 (optionally the additional mutation being a guanine); and/or (ii) position 106,330,066 on human chromosome 14 (optionally the additional mutation being a guanine).

Optionally the plurality comprises a human JH5 gene variant of SEQ ID NO: 1, wherein the variant comprises a cytosine at a position corresponding to position 106,330,027 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the plurality comprises a human JH5 gene variant of SEQ ID NO: 1, wherein the variant comprises an adenine at a position corresponding to position 106,330,024 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the plurality comprises a human JH5 gene variant of SEQ ID NO: 1, wherein the variant comprises a thymine at a position corresponding to position 106,330,032 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the plurality comprises a human JH5 gene variant of SEQ ID NO: 1, wherein the variant comprises a thymine at a position corresponding to position 106,330,041 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the plurality comprises a human JH5 gene variant of SEQ ID NO: 1, wherein the variant comprises an adenine or thymine at a position corresponding to position 106,330,063 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the variant comprises additionally a mutation at a position corresponding to position 106,330,071 on human chromosome 14 (optionally the additional mutation being a guanine).

Optionally the plurality comprises a human JH5 gene variant of SEQ ID NO: 1,wherein the variant comprises a cytosine at a position corresponding to position 106,330,062 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 1.

Optionally the genome comprises SEQ ID NO:1; optionally in cis at the same Ig locus as one, two or more of the variants.

JH6

An embodiment provides a vertebrate, cell or population of the invention whose genome comprises a plurality of JH6 gene segments, wherein the plurality comprises a human JH6 gene variant of SEQ ID NO: 2, wherein the variant comprises a nucleotide mutation at one or more positions corresponding to positions

106,329,411 106.329.413 106.329.414 106,329,417 106,329,419 106,329,426 106,329,434 106,329,435, and 106,329,468 on human chromosome 14.

Optionally the genome of the vertebrate, cell or population comprises a plurality of JH6 gene segments, wherein the plurality comprises a human JH6 gene variant of SEQ ID NO: 2, wherein the variant comprises a guanine at a position corresponding to position 106,329,435 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 2.

Optionally the variant comprises additionally a mutation at a position corresponding to (i) position 106,329,468 on human chromosome 14 (optionally the additional mutation being a guanine); (ii) position 106,329,419 on human chromosome 14 (optionally the additional mutation being an adenine); (iii) position 106,329,434 on human chromosome 14 (optionally the additional mutation being a cytosine) and/or position 106,329,414 on human chromosome 14 (optionally the additional mutation being a guanine); (iv) position 106,329,426 on human chromosome 14 (optionally the additional mutation being an adenine); (v) position 106,329,413 on human chromosome 14 (optionally the additional mutation being an adenine); (vi) position 106,329,417 on human chromosome 14 (optionally the additional mutation being a thymine); (vii) position 106,329,411 on human chromosome 14 (optionally the additional mutation being a thymine); (viii) position 106,329,451 on human chromosome 14 (optionally the additional mutation being an adenine); (ix) position 106,329,452 on human chromosome 14 (optionally the additional mutation being a cytosine); and/or (x) position 106,329,453 on human chromosome 14 (optionally the additional mutation being a cytosine).

Optionally the variant comprises additionally mutations at positions corresponding to position 106.329.451 on human chromosome 14, the additional mutation being an adenine; position 106.329.452 on human chromosome 14, the additional mutation being a cytosine; and position 106.329.453 on human chromosome 14, the additional mutation being a cytosine.

The vertebrate, cell or population optionally comprises a plurality of JH6 gene segments, wherein the plurality comprises a human JH6 gene variant of SEQ ID NO: 2, wherein the variant comprises a guanine at a position corresponding to position 106,329,468 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 2.

Optionally the variant comprises additionally a mutation at a position corresponding to position 106,329,435 on human chromosome 14 (optionally the additional mutation being a guanine).

Optionally the vertebrate, cell or population comprises a plurality of JH6 gene segments, wherein the plurality comprises a human JH6 gene variant of SEQ ID NO: 2, wherein the variant comprises a thymine at a position corresponding to position 106,329,417 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 2.

Optionally the variant comprises additionally a mutation at a position corresponding to position 106,329,435 on human chromosome 14 (optionally the additional mutation being a guanine).

Optionally the vertebrate, cell or population comprises a plurality of JH6 gene segments, wherein the plurality comprises a human JH6 gene variant of SEQ ID NO: 2, wherein the variant comprises a cytosine at a position corresponding to position 106,329,434 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 2.

Optionally the variant comprises additionally a mutation at a position corresponding to (i) position 106,329,414 on human chromosome 14 (optionally the additional mutation being a guanine); and/or (ii) position 106,329,435 on human chromosome 14 (optionally the additional mutation being a guanine).

Optionally the vertebrate, cell or population comprises a plurality of JH6 gene segments, wherein the plurality comprises a human JH6 gene variant of SEQ ID NO: 2, wherein the variant comprises a thymine at a position corresponding to position 106,329,411 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 2.

Optionally the variant comprises additionally a mutation at a position corresponding to position 106,329,435 on human chromosome 14 (optionally the additional mutation being a guanine).

Optionally the vertebrate, cell or population comprises a plurality of JH6 gene segments, wherein the plurality comprises a human JH6 gene variant that is an antisense sequence of a variant described above.

Optionally the genome comprises SEQ ID NO:2; optionally cis at the same Ig locus as one, two or more of the JH6 variants.

JH2

An embodiment provides a vertebrate, cell or population of the invention whose genome comprises a plurality of JH2 gene segments, wherein the plurality comprises a human JH2 gene variant of SEQ ID NO: 3, wherein the variant comprises a nucleotide mutation at one or more positions corresponding to positions

106,331,455 106,331,453, and 106,331,409 on human chromosome 14.

Optionally the vertebrate, cell or population comprises said plurality of JH2 gene segments, wherein the plurality comprises a human JH2 gene variant of SEQ ID NO: 3, wherein the variant comprises a guanine at a position corresponding to position 106,331,455 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 3.

Optionally the variant comprises additionally a mutation at a position corresponding to (i) position 106,331,453 on human chromosome 14 (optionally the additional mutation being an adenine); and/or (ii) position 106,331,409 on human chromosome 14 (optionally the additional mutation being an adenine); (iii) position 106,329,434 on human chromosome 14 (optionally the additional mutation being an adenine).

Optionally the vertebrate, cell or population comprises a plurality of JH2 gene segments, wherein the plurality comprises a human JH2 gene variant of SEQ ID NO: 3, wherein the variant comprises an adenine at a position corresponding to position 106,331,453 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 3.

Optionally the variant comprises additionally a mutation at a position corresponding to position 106,331,409 on human chromosome 14 (optionally the additional mutation being an adenine).

Optionally the vertebrate, cell or population comprises a plurality of JH2 gene segments, wherein the plurality comprises a human JH2 gene variant of SEQ ID NO: 3, wherein the variant comprises an adenine at a position corresponding to position 106,331,409 on human chromosome 14; and optionally no further mutation from the sequence of SEQ ID NO: 3.

Optionally the vertebrate, cell or population comprises a plurality of JH2 gene segments, wherein the plurality comprises a human JH2 gene variant that is an antisense sequence of a variant described above.

Optionally the genome comprises SEQ ID NO:3; optionally cis at the same Ig locus as one, two or more of the JH2 variants.

Optionally the vertebrate, cell or population genome comprises two or more different JH gene segments selected from SEQ ID NOs: 1 to 3 and variants described above; optionally wherein said JH gene segments are cis at the same immunoglobulin Ig locus.

Multiple Human D Gene Segment Variants

A specific application of this configuration is the provision of multiple human D gene segments as follows (as set out in numbered clauses, starting at clause number 154).

-   154. A non-human vertebrate (e.g., a mouse or rat) or a non-human     vertebrate cell (e.g., an ES cell or a B-cell) having a genome     comprising at least 3 human D gene segments of the same type (e.g.,     D2-2 gene segments), wherein at least two of the human D gene     segments are variants that are not identical to each other (e.g.,     D2-2ref and D2-2a).     -   In an example of any aspect of the sixth configuration of the         invention (V, D, J or C), one or more or all of the variants are         naturally-occurring human gene segments.     -   In an example of any aspect of the sixth configuration of the         invention (V, D, J or C), one or more of the variants may be a         synthetic variant of a human gene segment. -   155. A non-human vertebrate (e.g., a mouse or rat) or a non-human     vertebrate cell (e.g., an ES cell or a B-cell) having a genome     comprising at least 2 different non-endogenous D gene segments of     the same type (e.g., D2-2ref and D2-2a) cis at the same Ig locus. -   156. A non-human vertebrate (e.g., a mouse or rat) or a non-human     vertebrate cell (e.g., an ES cell or a B-cell) having a genome     comprising at least 2 different human D gene segments of the same     type (e.g., D2-2ref and D2-2a) trans at the same Ig locus; and     optionally a third human D gene segment (e.g., (e.g., D2-2ref, D2-2a     or D2-2b) of the same type, wherein the third D is cis with one of     said 2 different D gene segments. -   157. A population of non-human vertebrates (e.g., mice or rats)     comprising a repertoire of human D gene segments, wherein the     plurality comprises at least 2 different human D gene segments of     the same type (e.g., D2-2 gene segments), a first of said different     D gene segments (e.g., D2-2ref) is provided in the genome of a first     vertebrate of the population, and a second of said different D gene     segment (e.g., D2-2a) being provided in the genome of a second     vertebrate of the population, wherein the genome of the first     vertebrate does not comprise the second D gene segment. -   158. A non-human vertebrate (e.g., a mouse or rat) or a non-human     vertebrate cell (e.g., an ES cell or a B-cell) having a genome     comprising at least 2 different non-endogenous D gene segments of     the same type (e.g., human D2-2 gene segments), wherein the D gene     segments are derived from the genome sequence of different human     individuals that are not genetically related over at least 3     generations. -   159. A method of enhancing the human immunoglobulin gene diversity     of a non-human vertebrate (e.g., a mouse or rat), the method     comprising providing the vertebrate with a genome comprising at     least 3 human D gene segments of the same type (e.g., D2-2 gene     segments), wherein at least two of the human D gene segments are     variants that are not identical to each other (e.g., D2-2ref and     D2-2a). -   160. A method of enhancing the immunoglobulin gene diversity of a     non-human vertebrate (e.g., a mouse or rat), the method comprising     providing the vertebrate with a genome comprising at least 2     different non-endogenous D gene segments of the same type (e.g.,     human D2-2 gene segments) cis at the same Ig locus. -   161. A method of enhancing the immunoglobulin gene diversity of a     non-human vertebrate (e.g., a mouse or rat), the method comprising     providing the vertebrate with a genome comprising at least 2     different human D gene segments of the same type (e.g., D2-2ref and     D2-2a) trans at the same Ig locus; and optionally a third human D     gene segment (e.g., D2-2ref, D2-2a or D2-2b) of the same type,     wherein the third D is cis with one of said 2 different D gene     segments. -   162. A method of providing an enhanced human immunoglobulin D gene     segment repertoire, the method comprising providing a population of     non-human vertebrates (e.g., a mouse or rat) comprising a repertoire     of human D gene segments, wherein the method comprises providing at     least 2 different human D gene segments of the same type (e.g.,     D2-2ref and D2-2a), wherein a first of said different D gene     segments is provided in the genome of a first vertebrate of the     population, and a second of said different D gene segments is     provided in the genome of a second vertebrate of the population,     wherein the genome of the first vertebrate does not comprise the     second D gene segment. -   163. A method of enhancing the human immunoglobulin gene diversity     of a non-human vertebrate (e.g., a mouse or rat), the method     comprising providing the vertebrate with a genome comprising at     least 2 different non-endogenous D gene segments of the same type     (e.g., D2-2ref and D2-2a), wherein the D gene segments are derived     from the genome sequence of different human individuals that are not     genetically related over at least 3 generations. -   164. The vertebrate or cell of clause 154, 156 or 158, or the method     of clause 159, 161 or 163, wherein at least 2 or 3 of said different     gene segments are provided cis at the same Ig locus in said genome. -   165. The vertebrate or cell of clause 154, 155 or 156, or the method     of any one of clauses 159 to 162 and 164, wherein the D gene     segments are derived from the genome sequence of different human     individuals that are not genetically related over at least 3     generations. -   166. The vertebrate or cell of any one of clauses 154 to 157, or the     method of any one of clauses 159 to 162 and 165, wherein the D gene     segments are derived from the genome sequence of two or more     different human individuals; optionally wherein the different human     individuals are from different human populations. -   167. The vertebrate, cell or method of clause 166, wherein the     individuals are not genetically related. -   168. The vertebrate, cell or method of any one of clauses 154 to     167, wherein at least one of the different D segments is a synthetic     mutant of a human germline D gene segment. -   169. A method of enhancing the human immunoglobulin gene diversity     of a non-human vertebrate (e.g., a mouse or rat), the method     comprising providing the vertebrate with a genome comprising at     least 2 human D gene segments of the same type (e.g., D2-2ref and     D2-2a), wherein the D gene segments are derived from the genome     sequence of different human individuals that are not genetically     related over at least 3 generations; optionally wherein at least 2     or 3 of said different gene segments are provided at the same IgH     locus in said genome. -   170. The vertebrate or cell of any one of clauses 154 to 158 and 164     to 168, wherein the genome comprises a substantially complete     functional repertoire of human D gene segment types supplemented     with one, two or more variant human D gene segments, wherein said     substantially complete functional repertoire and the supplementary D     gene segments are not found together in the germline genome of a     human individual. -   171. The population of clause 157, wherein the population comprises     a substantially complete functional repertoire of human D gene     segment types supplemented with one, two or more variant human D     gene segments, wherein said substantially complete functional     repertoire and the supplementary D gene segments are not found     together in the germline genome of a human individual. -   172. A non-human vertebrate (e.g., a mouse or rat) or a non-human     cell (e.g., an ES cell or a B-cell) having a genome comprising a     substantially complete functional repertoire of human D gene segment     types supplemented with one, two or more variant human D gene     segments, wherein said substantially complete functional repertoire     and the supplementary D gene segments are not found together in the     germline genome of a human individual. -   173. A population of non-human vertebrates (e.g., mice or rats)     comprising a substantially complete functional repertoire of human     JH gene segment types supplemented with one, two or more variant     human D gene segments, wherein said substantially complete     functional repertoire and the supplementary D gene segments are not     found together in the germline genome of a human individual. -   174. The vertebrate or cell of clause 172 or the population of     clause 173, comprising first and second D gene segments selected     from D2-2ref and D2-2a; or D2-21 ref and D2-21a; or D3-10ref and     D3-10a; or D3-16ref and D3-16a; or D2-8ref and D2-8a; or D3-3ref and     D3-3a; or D4-23ref and D4-23a; or D6-13ref and D6-13a; or D3-9ref     and D3-9a; or D4-4ref and D4-4a; or D7-27ref and D7-27a;     -   Optionally wherein the first and/or second D gene segment is         present in two or more copies.     -   For example, there are provided two or three copies of the first         gene segment, optionally with one, two or three copies of the         second gene segment. Copies can be arranged in cis or trans. -   175. The vertebrate, cell or population of clause 174, comprising     human gene segments D2-2ref and D2-2a; and D3-3ref and D3-3a; and     optionally also D2-15.     -   In an example, the vertebrate, cell or population comprises one         or more D segments selected from human D3-3, D2-15, D3-9; D4-17;         D3-10; D2-2; D5-24; D6-19; D3-22; D6-13; D5-12; D1-26; D1-20;         D5-18; D3-16; D2-21; D1-14; D7-27; D1-1; D6-25; D2-14 and D4-23         (e.g., selected from D3-9*01; D4-17*01; D3-10*01; D2-2*02;         D5-24*01; D6-19*01; D3-22*01; D6-13*01; D5-12*01; D1-26*01;         D1-20*01; D5-18*01; D3-16*02; D2-21*02; D1-14*01; D7-27*02;         D1-1*01; D6-25*01; D2-15*01; and D4-23*01), together with the         reference sequence(s) of said selected segment(s). These were         found in variable domains having a HCDR3 length of at least 20         amino acids (see examples herein). -   176. A non-human vertebrate or vertebrate cell according to clause     155, comprising a genome that comprises VH, D and JH gene     repertoires comprising human gene segments, the D gene repertoire     comprising one or more of     -   a plurality of D2-2 gene segments provided by at least 2         different D2-2 gene segments in cis at the same Ig locus in said         genome;     -   a plurality of D2-21 gene segments provided by at least 2         different D2-21 gene segments in cis at the same Ig locus in         said genome;     -   a plurality of D3-10 gene segments provided by at least 2         different D3-10 gene segments in cis at the same Ig locus in         said genome;     -   a plurality of D3-16 gene segments provided by at least 2         different D3-16 gene segments in cis at the same Ig locus in         said genome;     -   a plurality of D2-8 gene segments provided by at least 2         different D2-8 gene segments in cis at the same Ig locus in said         genome;     -   a plurality of D3-3 gene segments provided by at least 2         different D3-3 gene segments in cis at the same Ig locus in said         genome;     -   a plurality of D4-23 gene segments provided by at least 2         different D4-23 gene segments in cis at the same Ig locus in         said genome;     -   a plurality of D6-13 gene segments provided by at least 2         different D6-13 gene segments in cis at the same Ig locus in         said genome;     -   a plurality of D3-9 gene segments provided by at least 2         different D3-9 gene segments in cis at the same Ig locus in said         genome;     -   a plurality of D4-4 gene segments provided by at least 2         different D4-4 gene segments in cis at the same Ig locus in said         genome; and     -   a plurality of D7-27 gene segments provided by at least 2         different D7-27 gene segments in cis at the same Ig locus in         said genome;     -   optionally wherein the D gene segments are derived from the         genome sequence of two or more different human individuals. -   177. A non-human vertebrate or vertebrate cell according to clause     155, comprising a genome that comprises VH, D and JH gene     repertoires comprising human gene segments, the D gene repertoire     comprising one or more of     -   a plurality of D2-2 gene segments provided by at least 2         different D2-2 gene segments in trans in said genome;     -   a plurality of D2-21 gene segments provided by at least 2         different D2-21 gene segments in trans in said genome;     -   a plurality of D3-10 gene segments provided by at least 2         different D3-10 gene segments in trans in said genome;     -   a plurality of D3-16 gene segments provided by at least 2         different D3-16 gene segments in trans in said genome;     -   a plurality of D2-8 gene segments provided by at least 2         different D2-8 gene segments in trans in said genome;     -   a plurality of D3-3 gene segments provided by at least 2         different D3-3 gene segments in trans in said genome;     -   a plurality of D4-23 gene segments provided by at least 2         different D4-23 gene segments in trans in said genome;     -   a plurality of D6-13 gene segments provided by at least 2         different D6-13 gene segments in trans in said genome;     -   a plurality of D3-9 gene segments provided by at least 2         different D3-9 gene segments in trans in said genome;     -   a plurality of D4-4 gene segments provided by at least 2         different D4-4 gene segments in trans in said genome; and     -   a plurality of D7-27 gene segments provided by at least 2         different D7-27 gene segments in trans in said genome;     -   optionally wherein the D gene segments are derived from the         genome sequence of two or more different human individuals. -   178. A non-human vertebrate or vertebrate cell (optionally an ES     cell or B-cell), according to clause 154, comprising a genome that     comprises VH, D and JH gene repertoires comprising human gene     segments, the D gene repertoire comprising one or more of a     plurality of D2-2 gene segments provided by at least 3 different     D2-2 gene segments; a plurality of D2-21 gene segments provided by     at least 3 different D2-21 gene segments; a plurality of D3-10 gene     segments provided by at least 3 different D3-10 gene segments; a     plurality of D3-16 gene segments provided by at least 3 different     D3-16 gene segments; a plurality of D2-8 gene segments provided by     at least 3 different D2-8 gene segments; a plurality of D3-3 gene     segments provided by at least 3 different D3-3 gene segments; a     plurality of D4-23 gene segments provided by at least 3 different     D4-23 gene segments; a plurality of D6-13 gene segments provided by     at least 3 different D6-13 gene segments; a plurality of D3-9 gene     segments provided by at least 3 different D3-9 gene segments; a     plurality of D4-4 gene segments provided by at least 3 different     D4-4 gene segments; and a plurality of D7-27 gene segments provided     by at least 3 different D7-27 gene segments;     optionally wherein the D gene segments are derived from the genome     sequence of two or three different human individuals;     optionally wherein at least 2 or 3 of said different gene segments     are provided in cis at the same Ig locus in said genome. -   179. The vertebrate or cell of clause 176, 177 or 178, wherein the     different human individuals are from different human populations. -   180. The vertebrate or cell of any one of clauses 176 to 179,     wherein the individuals are not genetically related. -   181. The vertebrate or cell of any one of clauses 176 to 180,     wherein at least one of the different D segments is a synthetic     mutant of a human germline D gene segment. -   182. A non-human vertebrate or vertebrate cell (optionally an ES     cell or B-cell) according to clause 158, comprising a genome     comprising human VH, D and JH gene repertoires, the D gene     repertoire comprising of one or more of a plurality of D2-2 gene     segments provided by at least 2 different D2-2 gene; optionally in     cis in said genome;     -   a plurality of D2-21 gene segments provided by at least 2         different D2-21 gene; optionally in cis in said genome;     -   a plurality of D3-10 gene segments provided by at least 2         different D3-10 gene; optionally in cis in said genome;     -   a plurality of D3-16 gene segments provided by at least 2         different D3-16 gene; optionally in cis in said genome;     -   a plurality of D2-8 gene segments provided by at least 2         different D2-8 gene; optionally in cis in said genome;     -   a plurality of D3-3 gene segments provided by at least 2         different D3-3 gene; optionally in cis in said genome;     -   a plurality of D4-23 gene segments provided by at least 2         different D4-23 gene; optionally in cis in said genome;     -   a plurality of D6-13 gene segments provided by at least 2         different D6-13 gene; optionally in cis in said genome;     -   a plurality of D3-9 gene segments provided by at least 2         different D3-9 gene; optionally in cis in said genome;     -   a plurality of D4-4 gene segments provided by at least 2         different D4-4 gene; optionally in cis in said genome; and     -   a plurality of D7-27 gene segments provided by at least 2         different D7-27 gene; optionally in cis in said genome;     -   wherein the D gene segments are derived from the genome sequence         of different human individuals that are not genetically related         over at least 3 generations. -   183. The vertebrate or cell of clause 182, wherein the human     individuals are from different human populations. -   184. The vertebrate, cell or population of any one of clauses 154 to     183, wherein one or more of the D gene segments is a variant of a     human germline D gene segment, wherein the variant gene segment     encodes an amino acid sequence that differs by 1, 2 or 3 amino acids     from the corresponding amino acid sequence encoded by the human     germline D gene segment, provided in that said amino acid sequence     encoded by the variant does not include a stop codon when said     corresponding amino acid sequence does not include a stop codon.     -   Optionally, the variant and germline D gene segments encode the         respective amino acid sequences in reading frame 2 (IMGT         numbering). See Briney et al 2012. -   185. The vertebrate, cell or population of clause 184, wherein said     corresponding amino acid sequence encoded by the human germline D     gene segment is a hydrophilic or hydrophobic sequence (according to     J Mol. Biol. 1997 Jul. 25; 270(4):587-97; Corbett S J et al; Table     2). -   186. The vertebrate, cell or population of clause 184 or 185,     comprising said variant and said germline human D gene segments;     optionally wherein the variant and germline human D gene segments     are cis on the same chromosome. -   187. The vertebrate, cell or population of any one of clauses 184 to     186, wherein germline human D gene segment is a D2, D3, D5 or D6     family gene segment; optionally a D2-2, D2-15, D3-3, D3-9, D3-10,     D3-22, D5-5, D5-18, D6-6, D6-13, D6-19 gene segment.     -   These D segments are usable in all three reading frames.     -   Optionally a variant of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of         these human germline D gene segments is used. -   188. The vertebrate, cell or population of any one of clauses 154 to     187, comprising a plurality of D2-2 gene segments, wherein the     plurality comprises D2-2 gene segments that vary from each other at     one or more nucleotide positions corresponding to positions     106,382,687 and 106,382,711     -   on human chromosome 14. -   189. The vertebrate, cell or population of clause 188, wherein the     plurality comprises a human D2-2 gene segment ((optionally two     copies and/or in homozygous state) comprising a thymine at a     position corresponding to position 106,382,687 on human chromosome     14; and optionally no further mutation from the sequence of D2-2ref. -   190. The vertebrate, cell or population of clause 188 or 189,     wherein the plurality comprises a human D2-2 gene segment comprising     a cytosine at a position corresponding to position 106,382,687 on     human chromosome 14; and optionally no further mutation from the     sequence of D2-2a. -   191. The vertebrate, cell or population of any one of clauses 188 to     190, wherein the plurality comprises a human D2-2 gene segment     comprising an adenine at a position corresponding to position     106,382,711 on human chromosome 14; and optionally no further     mutation from the sequence of D2-2b. -   192. The vertebrate, cell or population of any one of clauses 188 to     191, wherein the plurality comprises a human D2-2 gene segment     comprising an thymine at a position corresponding to position     106,382,711 on human chromosome 14; and optionally no further     mutation from the sequence of D2-2ref. -   193. The vertebrate, cell or population of any one of clauses 154 to     192, comprising a plurality of D7-27 gene segments, wherein the     plurality comprises D7-27 gene segments that vary from each other at     a nucleotide position corresponding to position 106,331,767 on human     chromosome 14. -   194. The vertebrate, cell or population of clause 193, wherein the     plurality comprises a human D7-27 gene segment (optionally two     copies and/or in homozygous state) comprising a cytosine at a     position corresponding to position 106,331,767 on human chromosome     14; and optionally no further mutation from the sequence of     D7-27ref. -   195. The vertebrate, cell or population of clause 193 or 194,     wherein the plurality comprises a human D7-27 gene segment     comprising a guanine at a position corresponding to position     106,331,767 on human chromosome 14; and optionally no further     mutation from the sequence of D7-27a. -   196. The vertebrate, cell or population of any one of clauses 154 to     195, comprising a plurality of D4-23 gene segments, wherein the     plurality comprises D4-23 gene segments that vary from each other at     a nucleotide position corresponding to position 106,350,740 on human     chromosome 14. -   197. The vertebrate, cell or population of clause 196, wherein the     plurality comprises a human D4-23 gene segment (optionally two     copies and/or in homozygous state) comprising an adenine at a     position corresponding to position 106,350,740 on human chromosome     14; and optionally no further mutation from the sequence of     D4-23ref. -   198. The vertebrate, cell or population of clause 196 or 197,     wherein the plurality comprises a human D4-23 gene segment     (optionally two copies and/or in homozygous state) comprising an     guanine at a position corresponding to position 106,350,740 on human     chromosome 14; and optionally no further mutation from the sequence     of D4-23a. -   199. The vertebrate, cell or population of any one of clauses 154 to     197, comprising a plurality of D2-21 gene segments, wherein the     plurality comprises D2-21 gene segments that vary from each other at     a nucleotide position corresponding to position 106,354,418 on human     chromosome 14. -   200. The vertebrate, cell or population of clause 199, wherein the     plurality comprises a human D2-21 gene segment (optionally two     copies and/or in homozygous state) comprising an adenine at a     position corresponding to position 106,354,418 on human chromosome     14; and optionally no further mutation from the sequence of D2-21     ref. -   201. The vertebrate, cell or population of clause 199 or 200,     wherein the plurality comprises a human D2-21 gene segment     (optionally two copies and/or in homozygous state) comprising a     guanine at a position corresponding to position 106,354,418 on human     chromosome 14; and optionally no further mutation from the sequence     of D2-21a. -   202. The vertebrate, cell or population of any one of clauses 154 to     201, comprising a plurality of D3-16 gene segments, wherein the     plurality comprises D3-16 gene segments that vary from each other at     a nucleotide position corresponding to position 106,354,418 on human     chromosome 14. -   203. The vertebrate, cell or population of clause 202, wherein the     plurality comprises a human D3-16 gene segment (optionally two     copies and/or in homozygous state) comprising a thymine at a     position corresponding to position 106,361,515 on human chromosome     14; and optionally no further mutation from the sequence of     D3-16ref. -   204. The vertebrate, cell or population of clause 202 or 203,     wherein the plurality comprises a human D3-16 gene segment     (optionally two copies and/or in homozygous state) comprising a     cytosine at a position corresponding to position 106,361,515 on     human chromosome 14; and optionally no further mutation from the     sequence of D3-16a. -   205. The vertebrate, cell or population of any one of clauses 154 to     204, comprising a plurality of D6-13 gene segments, wherein the     plurality comprises D6-13 gene segments that vary from each other at     a nucleotide position corresponding to position 106,367,013 on human     chromosome 14. -   206. The vertebrate, cell or population of clause 205, wherein the     plurality comprises a human D6-13 gene segment (optionally two     copies and/or in homozygous state) comprising a thymine at a     position corresponding to position 106,367,013 on human chromosome     14; and optionally no further mutation from the sequence of     D6-13ref. -   207. The vertebrate, cell or population of clause 205 or 206,     wherein the plurality comprises a human D6-13 gene segment     (optionally two copies and/or in homozygous state) comprising a     cytosine at a position corresponding to position 106,367,013 on     human chromosome 14; and optionally no further mutation from the     sequence of D6-13a. -   208. The vertebrate, cell or population of any one of clauses 154 to     207, comprising a plurality of D3-10 gene segments, wherein the     plurality comprises D3-10 gene segments that vary from each other at     one or more nucleotide positions corresponding to positions     -   106.370.370 and     -   106.370.371     -   on human chromosome 14. -   209. The vertebrate, cell or population of clause 208, wherein the     plurality comprises a human D3-10 gene segment (optionally two     copies and/or in homozygous state) comprising a thymine at a     position corresponding to position 106,370,370 on human chromosome     14; and optionally no further mutation from the sequence of     D3-10ref. -   210. The vertebrate, cell or population of clause 208 or 209,     wherein the plurality comprises a human D3-10 gene segment     (optionally two copies and/or in homozygous state) comprising a     cytosine at a position corresponding to position 106,370,370 on     human chromosome 14; and optionally no further mutation from the     sequence of D3-10a. -   211. The vertebrate, cell or population of clause 208, 209 or 210     wherein the plurality comprises a human D3-10 gene segment     (optionally two copies and/or in homozygous state) comprising an     adenine at a position corresponding to position 106,370,371 on human     chromosome 14; and optionally no further mutation from the sequence     of D3-10ref. -   212. The vertebrate, cell or population of any one of clauses 208 to     211, wherein the plurality comprises a human D3-10 gene segment     (optionally two copies and/or in homozygous state) comprising a     guanine at a position corresponding to position 106,370,371 on human     chromosome 14; and optionally no further mutation from the sequence     of D3-10b. -   213. The vertebrate, cell or population of any one of clauses 154 to     212, comprising a plurality of D3-9 gene segments, wherein the     plurality comprises D3-9 gene segments that vary from each other at     a nucleotide position corresponding to position 106,370,567 on human     chromosome 14. -   214. The vertebrate, cell or population of clause 213, wherein the     plurality comprises a human D3-9 gene segment (optionally two copies     and/or in homozygous state) comprising an adenine at a position     corresponding to position 106,370,567 on human chromosome 14; and     optionally no further mutation from the sequence of D3-9ref. -   215. The vertebrate, cell or population of clause 213 or 214,     wherein the plurality comprises a human D3-9 gene segment     (optionally two copies and/or in homozygous state) comprising a     thymine at a position corresponding to position 106,370,567 on human     chromosome 14; and optionally no further mutation from the sequence     of D3-9a. -   216. The vertebrate, cell or population of any one of clauses 154 to     215, comprising a plurality of D2-8 gene segments, wherein the     plurality comprises D2-8 gene segments that vary from each other at     one or more nucleotide positions corresponding to positions     -   106,373,085; 106,373,086 and 106,373,089 on human chromosome 14. -   217. The vertebrate, cell or population of clause 216, wherein the     plurality comprises a human D2-8 gene segment (optionally two copies     and/or in homozygous state) comprising a cytosine at a position     corresponding to position 106,373,085 on human chromosome 14. -   218. The vertebrate, cell or population of clause 216 or 217,     wherein the plurality comprises a human D2-8 gene segment     (optionally two copies and/or in homozygous state) comprising a     thymine at a position corresponding to position 106,373,085 on human     chromosome 14; and optionally no further mutation from the sequence     of D2-8b. -   219. The vertebrate, cell or population of clause 216, 217 or 218     wherein the plurality comprises a human D2-8 gene segment     (optionally two copies and/or in homozygous state) comprising a     cytosine at a position corresponding to position 106,373,086 on     human chromosome 14; and     -   optionally no further mutation from the sequence of D2-8ref. -   220. The vertebrate, cell or population of any one of clauses 216 to     219, wherein the plurality comprises a human D2-8 gene segment     comprising a thymine at a position corresponding to position     106,373,086 on human chromosome 14; and optionally no further     mutation from the sequence of D2-8ref. -   221. The vertebrate, cell or population of any one of clauses 154 to     220, comprising a plurality of D4-4 gene segments, wherein the     plurality comprises D4-4 gene segments that vary from each other at     one or more nucleotide positions corresponding to positions     -   106,379,086; and 106,379,089     -   on human chromosome 14. -   222. The vertebrate, cell or population of clause 221, wherein the     plurality comprises a D4-4 gene segment (optionally two copies     and/or in homozygous state) comprising a cytosine at a position     corresponding to position 106,379,086 on human chromosome 14; and     optionally no further mutation from the sequence of D4-4ref. -   223. The vertebrate, cell or population of clause 221 or 222,     wherein the plurality comprises a human D4-4 gene segment     (optionally two copies and/or in homozygous state) comprising a     thymine at a position corresponding to position 106,379,086 on human     chromosome 14; and optionally no further mutation from the sequence     of D4-4a. -   224. The vertebrate, cell or population of clause 221, 222 or 223     wherein the plurality comprises a human D4-4 gene segment     (optionally two copies and/or in homozygous state) comprising a     cytosine at a position corresponding to position 106,379,089 on     human chromosome 14; and optionally no further mutation from the     sequence of D4-4ref or a cytosine at a position corresponding to     position 106,379,086 on human chromosome 14. -   225. The vertebrate, cell or population of any one of clauses 221 to     224, wherein the plurality comprises a human D4-4 gene segment     (optionally two copies and/or in homozygous state) comprising a     thymine at a position corresponding to position 106,373,089 on human     chromosome 14; and optionally no further mutation from the sequence     of D4-4a. -   226. The vertebrate, cell or population of any one of clauses 154 to     225, comprising a plurality of D3-3 gene segments, wherein the     plurality comprises D3-3 gene segments that vary from each other at     one or more nucleotide positions corresponding to positions     -   106,380,241; and 106,380,246     -   on human chromosome 14. -   227. The vertebrate, cell or population of clause 226, wherein the     plurality comprises a D3-3 gene segment (optionally two copies     and/or in homozygous state) comprising a thymine at a position     corresponding to position 106,380,241 on human chromosome 14; and     optionally no further mutation from the sequence of D3-3ref. -   228. The vertebrate, cell or population of clause 226 or 227,     wherein the plurality comprises a human D3-3 gene segment     (optionally two copies and/or in homozygous state) comprising a     cytosine at a position corresponding to position 106,380,241 on     human chromosome 14; and optionally no further mutation from the     sequence of D3-3a. -   229. The vertebrate, cell or population of clause 226, 227 or 228     wherein the plurality comprises a human D3-3 gene segment     (optionally two copies and/or in homozygous state) comprising an     adenine at a position corresponding to position 106,380,246 on human     chromosome 14; and optionally no further mutation from the sequence     of D3-3ref. -   230. The vertebrate, cell or population of any one of clauses 226 to     229, wherein the plurality comprises a human D3-3 gene segment     (optionally two copies and/or in homozygous state) comprising a     thymine at a position corresponding to position 106,380,246 on human     chromosome 14; and optionally no further mutation from the sequence     of D3-3a.

Multiple Human JL Gene Segment Variants

A specific application of this configuration is the provision of multiple human JLgene segments (JK and/or JA) as follows (as set out in numbered paragraphs, starting at paragraph number 80).

-   80. A non-human vertebrate (e.g., a mouse or rat) or a non-human     vertebrate cell (e.g., an ES cell or a B-cell) having a genome     comprising at least 3 human JLgene segments of the same type (e.g.,     JK1), wherein at least two of the human JLgene segments are variants     that are not identical to each other. -   81. A non-human vertebrate (e.g., a mouse or rat) or a non-human     vertebrate cell (e.g., an ES cell or a B-cell) having a genome     comprising at least 2 different non-endogenous JL gene segments of     the same type (e.g., JK1) cis at the same Ig locus. -   82. A non-human vertebrate (e.g., a mouse or rat) or a non-human     vertebrate cell (e.g., an ES cell or a B-cell) having a genome     comprising at least 2 different human JLgene segments of the same     type (e.g., JK1) trans at the same Ig locus; and optionally a third     human JLgene segment of the same type, wherein the third JL is cis     with one of said 2 different JL gene segments. -   83. A population of non-human vertebrates (e.g., mice or rats)     comprising a repertoire of human JL gene segments, wherein the     plurality comprises at least 2 different human JL gene segments of     the same type (e.g., JK1), a first of said different JL gene     segments is provided in the genome of a first vertebrate of the     population, and a second of said different JLgene segments being     provided in the genome of a second vertebrate of the population,     wherein the genome of the first vertebrate does not comprise the     second JL gene segment. -   84. A non-human vertebrate (e.g., a mouse or rat) or a non-human     vertebrate cell (e.g., an ES cell or a B-cell) having a genome     comprising at least 2 different non-endogenous JL gene segments of     the same type (e.g., JK1), wherein the JL gene segments are derived     from the genome sequence of different human individuals that are not     genetically related over at least 3 generations. -   85. A method of enhancing the human immunoglobulin gene diversity of     a non-human vertebrate (e.g., a mouse or rat), the method comprising     providing the vertebrate with a genome comprising at least 3 human     JL gene segments of the same type (e.g., JK1), wherein at least two     of the human JL gene segments are variants that are not identical to     each other. -   86. A method of enhancing the immunoglobulin gene diversity of a     non-human vertebrate (e.g., a mouse or rat), the method comprising     providing the vertebrate with a genome comprising at least 2     different non-endogenous JLgene segments of the same type (e.g.,     JK1) cis at the same Ig locus. -   87. A method of enhancing the immunoglobulin gene diversity of a     non-human vertebrate (e.g., a mouse or rat), the method comprising     providing the vertebrate with a genome comprising at least 2     different human JLgene segments of the same type(e.g., JK1) trans at     the same Ig locus; and optionally a third human JLgene segment of     the same type, wherein the third JL is cis with one of said 2     different JL gene segments. -   88. A method of providing an enhanced human immunoglobulin JL gene     segment repertoire, the method comprising providing a population of     non-human vertebrates (e.g., a mouse or rat) comprising a repertoire     of human JL gene segments, wherein the method comprises providing at     least 2 different human JLgene segments of the same type (e.g.,     JK1), wherein a first of said different JLgene segments is provided     in the genome of a first vertebrate of the population, and a second     of said different JL gene segments is provided in the genome of a     second vertebrate of the population, wherein the genome of the first     vertebrate does not comprise the second JL gene segment. -   89. A method of enhancing the human immunoglobulin gene diversity of     a non-human vertebrate (e.g., a mouse or rat), the method comprising     providing the vertebrate with a genome comprising at least 2     different non-endogenous JLgene segments of the same type (e.g.,     JK1), wherein the JL gene segments are derived from the genome     sequence of different human individuals that are not genetically     related over at least 3 generations. -   90. The vertebrate or cell of paragraph 80, 82 or 84, or the method     of paragraph 85, 82 or 89, wherein at least 2 or 3 of said different     gene segments are provided cis at the same Ig locus in said genome. -   91. The vertebrate or cell of paragraph 80, 81 or 82, or the method     of paragraph 85, 86 or 87, wherein the JL gene segments are derived     from the genome sequence of different human individuals that are not     genetically related over at least 3 generations. -   92. The vertebrate or cell of paragraph 80, 81 or 82, or the method     of paragraph 85, 86 or 87, wherein the JL gene segments are derived     from the genome sequence of two or more different human individuals;     optionally wherein the different human individuals are from     different human populations. -   93. The vertebrate, cell or method of paragraph 92, wherein the     individuals are not genetically related. -   94. The vertebrate, cell or method of any one of paragraphs 80 to     93, wherein at least one of the different JL segments is a synthetic     mutant of a human germline JL gene segment. -   95. A method of enhancing the human immunoglobulin gene diversity of     a non-human vertebrate (e.g., a mouse or rat), the method comprising     providing the vertebrate with a genome comprising at least 2 human     JL gene segments of the same type (e.g., JK1),

wherein the JL gene segments are derived from the genome sequence of different human individuals that are not genetically related over at least 3 generations; optionally wherein at least 2 or 3 of said different gene segments are provided at the same IgL locus in said genome.

-   96. The vertebrate or cell of any one of paragraphs paragraph 80 to     82 and 84, wherein the genome comprises a substantially complete     functional repertoire of human JK and/or JA gene segment types     supplemented with one, two or more human JK and/or JA gene segments     respectively, wherein said substantially complete functional     repertoire and the supplementary gene segments are not found     together in the germline genome of a human individual. -   97. The population of paragraph 83, wherein the population comprises     a substantially complete functional repertoire of human JL gene     segment types supplemented with one, two or more human JK and/or JA     gene segments respectively, wherein said substantially complete     functional repertoire and the supplementary gene segments are not     found together in the germline genome of a human individual. -   98. A non-human vertebrate (e.g., a mouse or rat) or a non-human     cell (e.g., an ES cell or a B-cell) having a genome comprising a     substantially complete functional repertoire of human JK and/or JA     gene segment types supplemented with one, two or more human JK     and/or JA gene segments respectively, wherein said substantially     complete functional repertoire and the supplementary gene segments     are not found together in the germline genome of a human individual. -   99. A population of non-human vertebrates (e.g., mice or rats)     comprising a substantially complete functional repertoire of human     JK and/or JA gene segment types supplemented with one, two or more     human JK and/or JA gene segments respectively, wherein said     substantially complete functional repertoire and the supplementary     gene segments are not found together in the germline genome of a     human individual. -   100. A non-human vertebrate or vertebrate cell according to     paragraph 81, comprising a genome that comprises VL and JL gene     repertoires comprising human gene segments, the JL gene repertoire     comprising     -   a plurality of human JK1 gene segments provided by at least 2         different human JK1 gene segments in cis at the same Ig locus in         said genome;     -   a plurality of human JK2 gene segments provided by at least 2         different human JK1 gene segments in cis at the same Ig locus in         said genome;     -   a plurality of human JK3 gene segments provided by at least 2         different human JK1 gene segments in cis at the same Ig locus in         said genome;     -   a plurality of human JK4 gene segments provided by at least 2         different human JK1 gene segments in cis at the same Ig locus in         said genome;     -   a plurality of human JK5 gene segments provided by at least 2         different human JK1 gene segments in cis at the same Ig locus in         said genome;     -   a plurality of human JA1 gene segments provided by at least 2         different human JA1 gene segments in cis at the same Ig locus in         said genome;     -   a plurality of human JA2 gene segments provided by at least 2         different human JA2 gene segments in cis at the same Ig locus in         said genome;     -   a plurality of human JA3 gene segments provided by at least 2         different human JA3 gene segments in cis at the same Ig locus in         said genome;     -   a plurality of human JA4 gene segments provided by at least 2         different human JA4 gene segments in cis at the same Ig locus in         said genome;     -   a plurality of human JA5 gene segments provided by at least 2         different human JA5 gene segments in cis at the same Ig locus in         said genome;     -   a plurality of human JA6 gene segments provided by at least 2         different human JA6 gene segments in cis at the same Ig locus in         said genome; or     -   a plurality of human JA7 gene segments provided by at least 2         different human JA7 gene segments in cis at the same Ig locus in         said genome;     -   optionally wherein the JLgene segments are derived from the         genome sequence of two or more different human individuals. -   101. A non-human vertebrate or vertebrate cell (optionally an ES     cell or B-cell), according to paragraph 80, comprising a genome that     comprises VL and JL gene repertoires comprising human gene segments,     the JL gene repertoire comprising     -   a plurality of human JK1 gene segments provided by at least 3         (e.g., 3, 4, 5, 6, or 7) different human JK1 gene segments;     -   a plurality of human JK2 gene segments provided by at least 3         (e.g., 3, 4, 5, 6, or 7) different human JK1 gene segments;     -   a plurality of human JK3 gene segments provided by at least 3         (e.g., 3, 4, 5, 6, or 7) different human JK1 gene segments;     -   a plurality of human JK4 gene segments provided by at least 3         (e.g., 3, 4, 5, 6, or 7) different human JK1 gene segments;     -   a plurality of human JK5 gene segments provided by at least 3         (e.g., 3, 4, 5, 6, or 7) different human JK1 gene segments;     -   a plurality of human JA1 gene segments provided by at least 3         (e.g., 3, 4, 5, 6, or 7) different human JA1 gene segments;     -   a plurality of human JA2 gene segments provided by at least 3         (e.g., 3, 4, 5, 6, or 7) different human JA2 gene segments;     -   a plurality of human JA3 gene segments provided by at least 3         (e.g., 3, 4, 5, 6, or 7) different human JA3 gene segments;     -   a plurality of human JA4 gene segments provided by at least 3         (e.g., 3, 4, 5, 6, or 7) different human JA4 gene segments;     -   a plurality of human JA5 gene segments provided by at least 3         (e.g., 3, 4, 5, 6, or 7) different human JA5 gene segments;     -   a plurality of human JA6 gene segments provided by at least 3         (e.g., 3, 4, 5, 6, or 7) different human JA6 gene segments; or     -   a plurality of human JA7 gene segments provided by at least 3         (e.g., 3, 4, 5, 6, or 7) different human JA7 gene segments;     -   optionally wherein the JLgene segments are derived from the         genome sequence of two or three     -   different human individuals;     -   optionally wherein at least 2 or 3 of said different gene         segments are provided in cis at the same Ig locus in said         genome. -   102. The vertebrate or cell of paragraph 104 or 105, wherein the     different human individuals are from different human populations. -   103. The vertebrate or cell of any one of paragraphs 104 to 106,     wherein the individuals are not genetically related. -   104. The vertebrate or cell of any one of paragraphs 104 to 107,     wherein at least one of the different JL segments is a synthetic     mutant of a human germline JL gene segment. -   105. A non-human vertebrate or vertebrate cell (optionally an ES     cell or B-cell) according to paragraph 84, comprising a genome     comprising human VL and JL gene repertoires, the JL gene repertoire     comprising     -   a plurality of human JK1 gene segments provided by at least 2         different human JK1 gene segments, optionally in cis at the same         Ig locus in said genome;     -   a plurality of human JK2 gene segments provided by at least 2         different human JK1 gene segments, optionally in cis at the same         Ig locus in said genome;     -   a plurality of human JK3 gene segments provided by at least 2         different human JK1 gene segments, optionally in cis at the same         Ig locus in said genome;     -   a plurality of human JK4 gene segments provided by at least 2         different human JK1 gene segments, optionally in cis at the same         Ig locus in said genome;     -   a plurality of human JK5 gene segments provided by at least 2         different human JK1 gene segments, optionally in cis at the same         Ig locus in said genome;     -   a plurality of human JA1 gene segments provided by at least 2         different human JA1 gene segments, optionally in cis at the same         Ig locus in said genome;     -   a plurality of human JA2 gene segments provided by at least 2         different human JA2 gene segments, optionally in cis at the same         Ig locus in said genome;     -   a plurality of human JA3 gene segments provided by at least 2         different human JA3 gene segments, optionally in cis at the same         Ig locus in said genome;     -   a plurality of human JA4 gene segments provided by at least 2         different human JA4 gene segments, optionally in cis at the same         Ig locus in said genome;     -   a plurality of human JA5 gene segments provided by at least 2         different human JA5 gene segments, optionally in cis at the same         Ig locus in said genome;     -   a plurality of human JA6 gene segments provided by at least 2         different human JA6 gene segments, optionally in cis at the same         Ig locus in said genome; or a plurality of human JA7 gene         segments provided by at least 2 different human JA7 gene         segments, optionally in cis at the same Ig locus in said genome;     -   wherein the JL gene segments are derived from the genome         sequence of different human individuals that are not genetically         related over at least 3 generations. -   106. The vertebrate or cell of paragraph 109, wherein the human     individuals are from different human populations.     -   The skilled person will realise that standard molecular biology         techniques can be used to provide vectors comprising synthetic         combinations of immunoglobulin gene segments (e.g., V, D         and/or J) for use in the invention, such that the vectors can be         used to build a transgenic immunoglobulin locus (e.g., using         homologous recombination and/or recombinase mediated cassette         exchange as known in the art, e.g., see U.S. Pat. No. 7,501,552         (Medarex), U.S. Pat. No. 5,939,598 (Abgenix), U.S. Pat. No.         6,130,364 (Abgenix), WO02/066630 (Regeneron), WO2011004192         (Genome Research Limited), WO2009076464, WO2009143472 and         WO2010039900 (Ablexis), the disclosures of which are explicitly         incorporated herein. For example, such synthetic combinations of         gene segments can be made using standard recombineerinq         techniques in E. coli to construct BAC vectors harbouring the         synthetic combination prior to insertion in embryonic stem cells         using homologous recombination or RMCE (e.g., using cre/lox         site-specific recombination). Details of recombineering can be         found at www.genebridges.com and in EP1034260 and EP1204740 the         disclosures of which are explicitly incorporated herein.     -   In one embodiment, it is useful to bias the immune response of         the vertebrate (and thus resultant lead antibodies) to a         predetermined gene segment, e.g., one known to be commonly used         in natural human immune responses to antigens, such as antigens         of infectious disease pathogens. For example, VH1-69 is commonly         used to produce antibodies in humans against Influenza virus; it         is possible, therefore, to include two or more polymorphic DNA         versions of the VH segment VH1-69 in the locus of the invention.         The examples below illustrate how such a transgenic locus can be         constructed in which diversity is extended by extending the         VH1-69 gene segment repertoire based on naturally-occurring         VH1-69 polymorphic variants.     -   In one embodiment in any configuration of the invention, the         genome has been modified to prevent or reduce the expression of         fully-endogenous antibody. Examples of suitable techniques for         doing this can be found in PCT/GB2010/051122, U.S. Pat. No.         7,501,552, U.S. Pat. No. 6,673,986, U.S. Pat. No. 6,130,364,         WO2009/076464, EP1399559 and U.S. Pat. No. 6,586,251, the         disclosures of which are incorporated herein by reference. In         one embodiment, the non-human vertebrate VDJ region of the         endogenous heavy chain immunoglobulin locus, and optionally VJ         region of the endogenous light chain immunoglobulin loci (lambda         and/or kappa loci), have been inactivated. For example, all or         part of the non-human vertebrate VDJ region is inactivated by         inversion in the endogenous heavy chain immunoglobulin locus of         the mammal, optionally with the inverted region being moved         upstream or downstream of the endogenous Ig locus (see, e.g.,         WO2011004192, the disclosure of which is incorporated herein by         reference). For example, all or part of the non-human vertebrate         VJ region is inactivated by inversion in the endogenous kappa         chain immunoglobulin locus of the mammal, optionally with the         inverted region being moved upstream or downstream of the         endogenous Ig locus. For example, all or part of the non-human         vertebrate VJ region is inactivated by inversion in the         endogenous lambda chain immunoglobulin locus of the mammal,         optionally with the inverted region being moved upstream or         downstream of the endogenous Ig locus. In one embodiment the         endogenous heavy chain locus is inactivated in this way as is         one or both of the endogenous kappa and lambda loci.     -   Additionally or alternatively, the vertebrate has been generated         in a genetic background which prevents the production of mature         host B and T lymphocytes, optionally a RAG-1-deficient and/or         RAG-2 deficient background. See U.S. Pat. No. 5,859,301 for         techniques of generating RAG-1 deficient animals.     -   Thus, in one embodiment of any configuration or aspect of the         invention herein, endogenous heavy and light chain expression         has been inactivated.     -   In one embodiment each said locus constant region is a heavy         chain endogenous non-human vertebrate (optionally host mouse or         rat) constant region.     -   In one embodiment each said locus constant region is a light         chain endogenous non-human vertebrate (optionally host mouse or         rat) constant region.     -   The invention provides a monoclonal or polyclonal antibody         composition prepared by immunisation of at least one vertebrate         (e.g., mouse or rat) according to the invention, optionally         wherein the antigen is an antigen of an infectious disease         pathogen (e.g., a bacterial or viral pathogen antigen),         optionally wherein the same antigen is used to immunize all the         vertebrates; optionally wherein the antibody or antibodies are         IgG-type (e.g., IgG1).     -   The invention also provides a monoclonal or polyclonal antibody         mixture produced by the method of the invention or a derivative         antibody or mixture thereof, e.g., where one or more constant         region has been changed (e.g., replaced with a different         constant region such as a human constant region; or mutated to         enhance or ablate Fc effector function). In an aspect of the         invention, the monoclonal or polyclonal antibody mixture is         provided for therapy and/or prophylaxis of a disease or         condition in a human, e.g., for the treatment and/or prevention         of an infectious disease, wherein optionally wherein each         antibody binds an antigen of an infectious disease pathogen,         preferably the same antigen.     -   In an aspect of the invention, there is provided the use of an         isolated, monoclonal or polyclonal antibody according to the         invention, or a mutant or derivative antibody thereof in the         manufacture of a medicament for the treatment and/or prevention         of a disease or condition in a human, e.g., an infectious         disease, optionally wherein the infectious disease is a disease         caused by a bacterial or viral pathogen.     -   An example of a mutant antibody is one that bears up to 15 or 10         amino acid mutations in its variable regions relative to an         isolated antibody (e.g., IgG-type, such as IgG1-type, antibody)         obtainable or obtained by the method of the invention. An         example of a derivative is one that has been modified to replace         a constant region with a different constant region such as a         human constant region; or mutated to enhance or ablate Fc         effector function.     -   Examples of infectious diseases are diseases caused or mediated         by a bacterial or viral pathogen. For example, the infectious         disease is selected from the group consisting of a disease         caused by a pathogen selected from the group consisting of         Haemophilus influenza, E coli, Neisseria meningitidis, a herpes         family virus, cytomegalovirus (CMV), HIV and influenza virus.         Tailoring V(D)J Incorporation into Immunoglobin Loci for the         Generation of Antibodies Against Infectious Disease

The inventors realised that it would be desirable to provide for vertebrates, cells, methods etc for the production of therapeutic and/or prophylactic antibodies based on natural human immune responses to antigens, such as antigens of infectious disease pathogens. In this respect, the literature observes frequently used immunoglobulin gene segments to raise anti-infective responses in humans (Table 9).

In the various configurations, aspects, embodiments and examples above, the invention provides the skilled addressee with the possibility of choosing immunoglobulin gene segments in a way that tailors or biases the repertoire for application to generating antibodies to treat and/or prevent infectious diseases. The inventors have categorized the following groups of gene segments for use in the invention according to the desired application of resultant antibodies.

List A:

Immunoglobulin Gene Segments for Antibodies that Bind an Antigen Expressed by a Pathogen

-   (a) a VL gene segment selected from the group consisting of a VAII     gene family member, VAVII 4A, VAII 2.1, VAVII 4A, a VA1 gene family     member, a VA3gene family member, IGLV1S2, VA3-cML70, Ialh2, Ialyl,     Ia3h3, Kv325, a VKI gene family member, KI-15A (KL012), V°II family     member, a V°III family member, a VKI gene family member, KI-15A     (KL012), V°II A2 (optionally the A2a variant), VK A27 (Humkv325) and     a gene segment at least 80% identical thereto. -   (b) a VAgene segment selected from a VAII gene family member, VAVII     4A, VAII 2.1, VAVII 4A, a VA1 gene family member, a VA3gene family     member, IGLV1S2, VA3-cML70, Ialh2, Ialyl, Ia3h3 and a gene segment     at least 80% identical thereto. -   (c) a VK gene segment selected from Kv325, a VKI gene family member,     KI-15A (KL012), V°II family member, a VKIII family member, a VKI     gene family member, KI-15A (KL012), V°II A2 (optionally the A2a     variant), VK A27 (Humkv325) and a gene segment at least 80%     identical thereto. -   (d) a VH gene segment a VHIII gene family member (optionally, a     VHIIIa or VHIIIb family member), a VHIV gene family member, VHIII     9.1 (VH3-15), VHIII VH26 (VH3-23), VH3-21, LSG6.1, LSG12.1, DP77     (V3-21), VH H11, VH1GRR, ha3h2, VHI-ha1c, VHIII-VH2-1, VH4.18,     ha4h3, Hv1051, 71-2, Hv1f10, VH4.11, 71-4, VH251, VH1-69 and a gene     segment at least 80% identical thereto. -   (e) a JA gene segment selected from JA2, JA3 and a gene segment at     least 80% identical thereto. -   (f) a D gene segment selected from Dk1, Dxp>>1, Dn4r, D2r and a gene     segment at least 80% identical thereto.

List A1:

Immunoglobulin Gene Segments for Antibodies that Bind an Antigen Expressed by a Pathogen

-   (a) a VAgene segment selected from a VAII gene family member, VAVII     4A, VAII 2.1, VAVII 4A and a gene segment at least 80% identical     thereto. -   (b) a VK gene segment selected from a VKI gene family member, KI-15A     (KL012), V°II family member, a VKIII family member, a VKI gene     family member, KI-15A (KL012), V°II A2 (optionally the A2a variant),     VK A27 (Humkv325) and a gene segment at least 80% identical thereto. -   (c) a VH gene segment a VH3 gene family member (optionally, a VHIIIa     or VHIIIb family member), VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23),     VH3-21, LSG6.1, LSG12.1, DP77 (V3-21), VH H11 and a gene segment at     least 80% identical thereto. -   (d) a JA gene segment selected from JA2, JA3 and a gene segment at     least 80% identical thereto. -   (e) a JH gene segment selected from JH2, JH3, JH4 and a gene segment     at least 80% identical thereto.

List A1.1:

Immunoglobulin Gene Segments for Antibodies that Bind an Antigen Expressed by H Influenza

-   (a) a VAgene segment selected from a VAII gene family member, VAVII     4A, VAII 2.1, VAVII 4A and a gene segment at least 80% identical     thereto. -   (b) a VK gene segment selected from a V°II family member, a VKIII     family member, a VKI gene family member, KI-15A (KL012), V°II A2     (optionally the A2a variant), V°A27 (Humkv325) and a gene segment at     least 80% identical thereto. -   (c) a VH gene segment a VH3 gene family member (optionally, a VHIIIb     family member), VHIII 9.1 (VH3-15), VHIII VH26 (VH3-23), VH3-21,     LSG6.1, LSG12.1, DP77 (V3-21) and a gene segment at least 80%     identical thereto. -   (d) a JA gene segment selected from JA2, JA3 and a gene segment at     least 80% identical thereto.

List A1.2:

Immunoglobulin Gene Segments for Antibodies that Bind an Antigen Expressed by E. coli or Neisseria meningitidis

-   (a) a VH gene segment a VH3 gene family member (optionally a VHIIIa     or VHIIIb member), VHIII 9.1 (VH3-15), VH H11, VHIII VH26 (VH3-23) a     gene segment at least 80% identical thereto, e.g., VHIII 9.1© JH3;     or VH H11© JH4; or VHIII VH26© JH2. -   (b) a VK gene segment selected from a VKI gene family member, KI-15A     (KL012) and a gene segment at least 80% identical thereto. -   (c) a VAgene segment selected from a VAII gene family member, VAII     2.1 and a gene segment at least 80% identical thereto. -   (d) a JH gene segment selected from JH2, JH3, JH4 and a gene segment     at least 80% identical thereto.

A2:

Immunoglobulin Gene Segments for Antibodies that Bind an Antigen Expressed by a Viral Pathogen

-   (a) a VH gene segment selected from a VHIII gene family member, a     VHIV gene family member, VHIII-VH26 (VH3-23), VH1GRR, ha3h2,     VHI-ha1c1, VHIII-VH2-1, VH4.18, ha4h3, Hv1051, 71-2, Hv1f10, VH4.11,     71-4, VH251, VH1-69 and a gene segment at least 80% identical     thereto. -   (b) a VA gene segment selected from a VA1 gene family member, a     VA3gene family member, IGLV1S2, VA3-cML70, Ialh2, Ialyl, Ia3h3 and a     gene segment at least 80% identical thereto. -   (c) a Vk gene segment selected from Kv325 and a gene segment at     least 80% identical thereto. -   (d) a JH gene segment selected from JH3, JH5, JH6 and a gene segment     at least 80% identical thereto. -   (e) a D gene segment selected from Dk1, Dxp>>1, Dn4r, D2r and a gene     segment at least 80% identical thereto. -   (f) a JA gene segment selected from JA2, JA3 and a gene segment at     least 80% identical thereto.

A2.1:

Immunoglobulin Gene Segments for Antibodies that Bind an Antigen Expressed by Herpes Virus Family (e.g., VZV or HSV)

-   (a) a VH gene segment selected from a VHIII gene family member, a     VHIV gene family member, VHIII-VH26 (VH3-23), VH1GRR, ha3h2,     VHI-ha1c1, VHIII-VH2-1, VH4.18, ha4h3, and a gene segment at least     80% identical thereto. -   (b) a VA gene segment selected from a VA1 gene family member, a     VA3gene family member, IGLV1S2, VA3-cML70, Ialh2, Ialyl, Ia3h3 and a     gene segment at least 80% identical thereto. -   (c) a JH gene segment selected from JH3, JH5, JH6 and a gene segment     at least 80% identical thereto. -   (d) a D gene segment selected from Dk1, Dxp>>1, Dn4r, D2r and a gene     segment at least 80% identical thereto. -   (e) a JA gene segment selected from JA2, JA3 and a gene segment at     least 80% identical thereto.

A2.2:

Immunoglobulin Gene Segments for Antibodies that Bind an Antigen Expressed by CMV

-   (a) a VH gene segment selected from Hv1051 and a gene segment at     least 80% identical thereto. -   (b) a Vk gene segment selected from Kv325 and a gene segment at     least 80% identical thereto. A2.3:     Immunoglobulin Gene Segments for Antibodies that Bind an Antigen     Expressed by HIV -   (a) a VH gene segment selected from 71-2, Hv1f10, VH4.11, 71-4,     VH251, VH1-69 and a gene segment at least 80% identical thereto.

A2.4:

Immunoglobulin Gene Segments for Antibodies that Bind an Antigen Expressed by Influenza Virus

-   (a) a VH gene segment selected from VH1-69 and a gene segment at     least 80% identical thereto.

Thus,

Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease, one or more V, D and/or or all J gene segments used in any configuration, aspect, method, example or embodiment of the invention can be selected from List A1. Thus, for example in (a) of the first configuration of the invention, the recited heavy chain V gene segment is selected from the VH gene segments in List A, optionally with a D in that list.

Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease caused or mediated by a bacterial pathogen, one or more or all V, D and/or J gene segments used in any configuration, aspect, method, example or embodiment of the invention can be selected from List A1.

Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease caused or mediated by a viral pathogen, one or more or all V, D and/or J gene segments used in any configuration, aspect, method, example or embodiment of the invention can be selected from List A2.

Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease caused or mediated by H influenza, one or more or all V, D and/or J gene segments used in any configuration, aspect, method, example or embodiment of the invention can be selected from List A1.1.

Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease caused or mediated by E. coli or Neisseria meningitidis, one or more or all V, D and/or J gene segments used in any configuration, aspect, method, example or embodiment of the invention can be selected from List A1.2.

Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease caused or mediated by Herpes Virus Family (e.g., VZV or HSV), one or more or all V, D and/or J gene segments used in any configuration, aspect, method, example or embodiment of the invention can be selected from List A2.1.

Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease caused or mediated by CMV, one or more or all V, D and/or J gene segments used in any configuration, aspect, method, example or embodiment of the invention can be selected from List A2.2.

Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease caused or mediated by HIV, one or more or all V, D and/or J gene segments used in any configuration, aspect, method, example or embodiment of the invention can be selected from List A2.3.

Where one wishes to generate an antibody or antibody mixture to treat and/or prevent an infectious disease caused or mediated by Influenza Virus, one or more or all V, D and/or J gene segments used in any configuration, aspect, method, example or embodiment of the invention can be selected from List A2.4.

Optionally each VH segment in the locus of the invention is selected from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4.

Optionally each VL segment in the locus of the invention is selected from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4

Optionally each D segment in the locus of the invention is selected from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4.

Optionally each JL segment in the locus of the invention is selected from List A1, A2, A1.1, A1.2, A2.1, A2.2, A2.3 or A2.4.

Antibodies for Therapy & Prophylaxis of Patients of Specific Ancestry

The inventors, having undertaken the extensive Bioinformatics analysis exercise described herein, realised that the output of that analysis has made it possible to identify specific gene segments that are useful to produce antibody- and VH domain-based drugs that are tailored specifically to a patient's ancestry (i.e., genotype). That is, antibodies can be selected on the basis that they are made in vivo in a transgenic non-human vertebrate (e.g., mouse or rat with transgenic IgH loci) and particularly derived from gene segments that are relatively prevalent in members of the patient's population, i.e., from individuals of the same human ancestry. Since variant distributions differ across different populations (see Table 13), this presumably reflects the effects of evolution, adaptation and conservation of useful variant gene types in those populations. Thus, by tailoring the antibody-based drugs according to the invention, it is possible to match the drug to the population gene biases, thus with the aim of making better drugs for that specific population of humans. Better can, for example, mean more efficacious, better neutralizing, higher target antigen affinity, less immunogenic, less patient reactions to the drug etc. This can be determined empirically, as is standard in drug research and development processes.

Thus, the invention provides the following embodiments (numbered from clause 345 onwards):—

-   345. An isolated antibody for administration to a Chinese patient,     the antibody comprising a human heavy chain, the heavy chain     comprising a variable domain that is specific for an antigen and a     constant region, wherein the constant region is a human constant     region selected from a constant region (e.g., an IGHG constant     region) in Table 13 found in a Chinese population and with a     cumulative frequency of at least 1 or 5%; and wherein     -   (i) the variable domain is derived from the recombination of         said human gene segments in a non-human vertebrate (e.g., in a         mouse or a rat); and/or (ii) the variable domain comprises         non-human vertebrate (e.g., mouse or rat) AID-pattern mutations         and non-human vertebrate (e.g., mouse or rat) terminal         deoxynucleotidyl transferase (TdT)-pattern mutations.     -   In another embodiment, the invention provides     -   An isolated antibody for administration to a Chinese patient,         the antibody comprising a human heavy chain, the heavy chain         comprising a variable domain that is specific for an antigen and         a constant region, wherein the constant region is a human         constant region selected from a constant region (e.g., an IGHG         constant region) present in a Chinese population with a         cumulative frequency of at least 5%;, and wherein     -   (i) the variable domain is derived from the recombination of         said human gene segments in a non-human vertebrate (e.g., in a         mouse or a rat); and/or (ii) the variable domain comprises         non-human vertebrate (e.g., mouse or rat) AID-pattern mutations         and non-human vertebrate (e.g., mouse or rat) terminal         deoxynucleotidyl transferase (TdT)-pattern mutations.     -   In an example, the constant region is found in the 1000 Genomes         database. In an example, the constant region is found in Table         13. -   346. The antibody of clause 345 wherein the constant region is a     IGHG1a, IGHG2a, IGHG3a, IGHG3b or IGHG4a constant region. -   347. The antibody of clause 345 or 346, wherein the variable domain     is derived from the recombination of a human VH gene segment with a     human D gene segment and a human JH gene segment, the VH gene     segment being selected from a VH in Table 13 found in a Chinese     population and with a cumulative frequency of at least 5%.     -   In another embodiment, the invention provides     -   The antibody of clause 345 or 346, wherein the variable domain         is derived from the recombination of a human VH gene segment         with a human D gene segment and a human JH gene segment, the VH         gene segment being selected from a VH present in a Chinese         population with a cumulative frequency of at least 5%.     -   In an example, the gene segment is found in the 1000 Genomes         database. In an example, the gene segment is found in Table 13. -   348. The antibody of clause 345, 346 or 347, wherein the variable     domain is derived from the recombination of a human VH gene segment     with a human D gene segment and a human JH gene segment, the D gene     segment being selected from a D in Table 13 found in a Chinese     population and with a cumulative frequency of at least 5%.     -   In another embodiment, the invention provides     -   The antibody of clause 345, 346 or 347, wherein the variable         domain is derived from the recombination of a human VH gene         segment with a human D gene segment and a human JH gene segment,         the D gene segment being selected from a D present in a Chinese         population with a cumulative frequency of at least 5%.     -   In an example, the gene segment is found in the 1000 Genomes         database. In an example, the gene segment is found in Table 13. -   349. The antibody of clause 345, 346, 347 or 348 wherein the     variable domain is derived from the recombination of a human VH gene     segment with a human D gene segment and a human JH gene segment, the     JH gene segment being selected from a JH in Table 13 found in a     Chinese population and with a cumulative frequency of at least 5%.     -   In another embodiment, the invention provides     -   The antibody of clause 345, 346, 347 or 348 wherein the variable         domain is derived from the recombination of a human VH gene         segment with a human D gene segment and a human JH gene segment,         the JH gene segment being selected from a JH present in a         Chinese population with a cumulative frequency of at least 5%.     -   In an example, the gene segment is found in the 1000 Genomes         database. In an example, the gene segment is found in Table 13. -   350. An isolated VH domain identical to a variable domain as recited     in any one of clauses 347 to 349, optionally fused at its C-terminus     to a polypeptide (e.g., an antibody Fc).     -   In an embodiment, there is provided an isolated VH domain         identical to a variable domain as recited in any one of clauses         347 to 349 which is part of a conjugate, conjugated with a label         (e.g., for imaging in the patient) or a toxin (e.g., a         radioactive toxic payload, such as for cancer treatment in the         patient) or a half-life-extending moiety (e.g., PEG of human         serum albumin). -   351. A pharmaceutical composition comprising the antibody or     variable domain of any one of clauses 345 to 350 together with a     pharmaceutically-acceptable excipient, diluent or a medicament     (e.g., a further antigen-specific variable domain, antibody chain or     antibody). -   352. An isolated antibody for administration to a Chinese patient,     the antibody comprising a human heavy chain, the heavy chain     comprising a variable domain that is specific for an antigen and a     constant region, wherein the variable domain is derived from the     recombination of a human VH gene segment with a human D gene segment     and a human JH gene segment, the VH gene segment being selected from     a VH in Table 13 found in a Chinese population and with a cumulative     frequency of at least 5%; and wherein     -   (i) the variable domain is derived from the recombination of         said human gene segments in a non-human vertebrate (e.g., in a         mouse or a rat); and/or (ii) the variable domain comprises         non-human vertebrate (e.g., mouse or rat) AID-pattern mutations         and non-human vertebrate (e.g., mouse or rat) terminal         deoxynucleotidyl transferase (TdT)-pattern mutations.         -   In another embodiment, the invention provides         -   An isolated antibody for administration to a Chinese             patient, the antibody comprising a human heavy chain, the             heavy chain comprising a variable domain that is specific             for an antigen and a constant region, wherein the variable             domain is derived from the recombination of a human VH gene             segment with a human D gene segment and a human JH gene             segment, the VH gene segment being selected from a VH             present in a Chinese population with a cumulative frequency             of at least 5%; and wherein     -   (i) the variable domain is derived from the recombination of         said human gene segments in a non-human vertebrate (e.g., in a         mouse or a rat); and/or (ii) the variable domain comprises         non-human vertebrate (e.g., mouse or rat) AID-pattern mutations         and non-human vertebrate (e.g., mouse or rat) terminal         deoxynucleotidyl transferase (TdT)-pattern mutations. -   353. The antibody of clause 352, wherein the variable domain is     derived from the recombination of a human VH gene segment with a     human D gene segment and a human JH gene segment, the D gene segment     being selected from a D in Table 13 found in a Chinese population     and with a cumulative frequency of at least 5%.     -   In another embodiment, the invention provides     -   The antibody of clause 352, wherein the variable domain is         derived from the recombination of a human VH gene segment with a         human D gene segment and a human JH gene segment, the D gene         segment being selected from a D present in a Chinese population         with a cumulative frequency of at least 5%.     -   In an example, the gene segment is found in the 1000 Genomes         database. In an example, the gene segment is found in Table 13. -   354. The antibody of clause 352 or 353, wherein the variable domain     is derived from the recombination of a human VH gene segment with a     human D gene segment and a human JH gene segment, the JH gene     segment being selected from a JH in Table 13 found in a Chinese     population and with a cumulative frequency of at least 5%.     -   In another embodiment, the invention provides     -   The antibody of clause 352 or 353, wherein the variable domain         is derived from the recombination of a human VH gene segment         with a human D gene segment and a human JH gene segment, the JH         gene segment being selected from a JH present in a Chinese         population with a cumulative frequency of at least 5%.     -   In an example, the gene segment is found in the 1000 Genomes         database. In an example, the gene segment is found in Table 13. -   355. An isolated VH domain identical to a variable domain as recited     in any one of clauses 352 to 354, optionally fused at its C-terminus     to a polypeptide (e.g., an antibody Fc).     -   In an embodiment, there is provided a VH domain identical to a         variable domain as recited in any one of clauses 352 to 354         which is part of a conjugate, conjugated with a label (e.g., for         imaging in the patient) or a toxin (e.g., a radioactive toxic         payload, such as for cancer treatment in the patient) or a         half-life-extending moiety (e.g., PEG of human serum albumin). -   356. A pharmaceutical composition comprising the antibody or     variable domain of any one of clauses 352 to 355 together with a     pharmaceutically-acceptable excipient, diluent or a medicament     (e.g., a further antigen-specific variable domain, antibody chain or     antibody). -   357. An antibody heavy chain or VH domain (e.g., provided as part of     an antibody) for therapy and/or prophylaxis of a disease or medical     condition in a Chinese patient, wherein the heavy chain is a heavy     chain produced by the following steps (or is a copy of such a heavy     chain):—     -   (a) Selection of an antigen-specific antibody heavy chain or VH         domain from a non-human vertebrate (e.g., a mouse or a rat),         wherein the heavy chain or VH domain is derived from the         recombination of a human VH gene segment with a human D gene         segment and a human JH gene segment, the VH gene segment being         selected from a VH in Table 13 found in a Chinese population and         with a cumulative frequency of at least 5%;     -   (b) Optional humanization of the heavy chain by combining the         variable domain of the heavy chain with a human constant region;         or optional humanization of the selected VH domain by combining         with a human constant region.

In another embodiment, the invention provides

An antibody heavy chain or VH domain (e.g., provided as part of an antibody) for therapy and/or prophylaxis of a disease or medical condition in a Chinese patient, wherein the heavy chain is a heavy chain produced by the following steps (or is a copy of such a heavy chain):—

-   (a) Selection of an antigen-specific antibody heavy chain or VH     domain from a non-human vertebrate (e.g., a mouse or a rat), wherein     the heavy chain or VH domain is derived from the recombination of a     human VH gene segment with a human D gene segment and a human JH     gene segment, the VH gene segment being selected from a VH present     in a Chinese population with a cumulative frequency of at least 5%; -   (b) Optional humanization of the heavy chain by combining the     variable domain of the heavy chain with a human constant region; or     optional humanization of the selected VH domain by combining with a     human constant region.

In an example, the VH gene segment is found in the 1000 Genomes database. In an example, the gene segment is found in Table 13.

-   358. The antibody heavy chain or VH domain of clause 357, wherein     the human constant region is as recited in clause 345 or 346. -   359. An antibody heavy chain or VH domain as recited in clause 357     or 358 for use in a medicament for therapy and/or prophylaxis of a     disease or medical condition in a Chinese patient. -   360. A method of treating and/or preventing a disease or medical     condition in a Chinese patient, the method comprising administering     to the patient a therapeutically or prophylactically-effective     amount of the antibody heavy chain or VH domain as recited in clause     357 or 358. -   361. An isolated antibody for administration to a patient of     European, East Asian, West African, South Asian or Americas     ancestry, the antibody comprising a human heavy chain, the heavy     chain comprising a variable domain that is specific for an antigen     and a constant region, wherein the constant region is a human     constant region selected from a constant region (e.g., an IGHG     constant region) in Table 13 found in a population of European, East     Asian, West African, South Asian or Americas ancestry respectively     and with a cumulative frequency of at least 1 or 5%;, and wherein     -   (i) the variable domain is derived from the recombination of         said human gene segments in a non-human vertebrate (e.g., in a         mouse or a rat); or (ii) the variable domain comprises non-human         vertebrate (e.g., mouse or rat) AID-pattern mutations and         non-human vertebrate (e.g., mouse or rat) terminal         deoxynucleotidyl transferase (TdT)-pattern mutations.     -   In another embodiment, the invention provides     -   An isolated antibody for administration to a patient of         European, East Asian, West African, South Asian or Americas         ancestry, the antibody comprising a human heavy chain, the heavy         chain comprising a variable domain that is specific for an         antigen and a constant region, wherein the constant region is a         human constant region selected from a constant region (e.g., an         IGHG constant region) present in a population of European, East         Asian, West African, South Asian or Americas ancestry         respectively with a cumulative frequency of at least 1 or 5%;,         and wherein     -   (i) the variable domain is derived from the recombination of         said human gene segments in a non-human vertebrate (e.g., in a         mouse or a rat); or (ii) the variable domain comprises non-human         vertebrate (e.g., mouse or rat) AID-pattern mutations and         non-human vertebrate (e.g., mouse or rat) terminal         deoxynucleotidyl transferase (TdT)-pattern mutations.     -   In an example, the constant region is found in the 1000 Genomes         database. In an example, the constant region is found in Table         13. -   362. The antibody of clause 361 wherein the constant region is a     IGHG1a, IGHG2a, IGHG3a, IGHG3b or IGHG4a constant region and the     patient is of European ancestry. -   363. The antibody of clause 361 or 362, wherein the variable domain     is derived from the recombination of a human VH gene segment with a     human D gene segment and a human JH gene segment, the VH gene     segment being selected from a VH in Table 13 found in said     population and with a cumulative frequency of at least 1 or 5%.

In another embodiment, the invention provides

The antibody of clause 361 or 362, wherein the variable domain is derived from the recombination of a human VH gene segment with a human D gene segment and a human JH gene segment, the VH gene segment being selected from a VH present in a Chinese population with a cumulative frequency of at least 5%.

In an example, the gene segment is found in the 1000 Genomes database. In an example, the gene segment is found in Table 13.

-   364. The antibody of clause 361, 362 or 363, wherein the variable     domain is derived from the recombination of a human VH gene segment     with a human D gene segment and a human JH gene segment, the D gene     segment being selected from a D in Table 13 found in said population     and with a cumulative frequency of at least 1 or 5%.     -   In another embodiment, the invention provides     -   The antibody of clause 361, 362 or 363, wherein the variable         domain is derived from the recombination of a human VH gene         segment with a human D gene segment and a human JH gene segment,         the D gene segment being selected from a D present in a Chinese         population with a cumulative frequency of at least 5%.     -   In an example, the gene segment is found in the 1000 Genomes         database. In an example, the gene segment is found in Table 13. -   365. The antibody of clause 361, 362, 363 or 364 wherein the     variable domain is derived from the recombination of a human VH gene     segment with a human D gene segment and a human JH gene segment, the     JH gene segment being selected from a JH in Table 13 found in said     population and with a cumulative frequency of at least 1 or 5%.     -   In another embodiment, the invention provides     -   The antibody of clause 361, 362, 363 or 364 wherein the variable         domain is derived from the recombination of a human VH gene         segment with a human D gene segment and a human JH gene segment,         the JH gene segment being selected from a JH present in a         Chinese population with a cumulative frequency of at least 5%.     -   In an example, the gene segment is found in the 1000 Genomes         database. In an example, the gene segment is found in Table 13. -   366. An isolated VH domain identical to a variable domain as recited     in any one of clauses 363 to 365, optionally fused at its C-terminus     to a polypeptide (e.g., an antibody Fc). -   367. A pharmaceutical composition comprising the antibody or     variable domain of any one of clauses 361 to 366 together with a     pharmaceutically-acceptable excipient, diluent or a medicament     (e.g., a further antigen-specific variable domain, antibody chain or     antibody). -   368. An isolated antibody for administration to a patient of     European, East Asian, West African or Americas ancestry, the     antibody comprising a human heavy chain, the heavy chain comprising     a variable domain that is specific for an antigen and a constant     region, wherein the variable domain is derived from the     recombination of a human VH gene segment with a human D gene segment     and a human JH gene segment, the VH gene segment being selected from     a VH in Table 13 found in a population of European, East Asian, West     African, South Asian or Americas ancestry respectively and with a     cumulative frequency of at least 1 or 5%; and wherein     -   (i) the variable domain is derived from the recombination of         said human gene segments in a non-human vertebrate (e.g., in a         mouse or a rat); or (ii) the variable domain comprises non-human         vertebrate (e.g., mouse or rat) AID-pattern mutations and         non-human vertebrate (e.g., mouse or rat) terminal         deoxynucleotidyl transferase (TdT)-pattern mutations.         -   In another embodiment the invention provides:—         -   An isolated antibody for administration to a patient of             European, East Asian, West African or Americas ancestry, the             antibody comprising a human heavy chain, the heavy chain             comprising a variable domain that is specific for an antigen             and a constant region, wherein the variable domain is             derived from the recombination of a human VH gene segment             with a human D gene segment and a human JH gene segment, the             VH gene segment being selected from a VH present in a             population of European, East Asian, West African, South             Asian or Americas ancestry respectively with a cumulative             frequency of at least 1 or 5%; and wherein     -   (i) the variable domain is derived from the recombination of         said human gene segments in a non-human vertebrate (e.g., in a         mouse or a rat); or (ii) the variable domain comprises non-human         vertebrate (e.g., mouse or rat) AID-pattern mutations and         non-human vertebrate (e.g., mouse or rat) terminal         deoxynucleotidyl transferase (TdT)-pattern mutations.     -   In an example, the VH gene segment is found in the 1000 Genomes         database. In an example, the gene segment is found in Table 13. -   369. The antibody of clause 368, wherein the variable domain is     derived from the recombination of a human VH gene segment with a     human D gene segment and a human JH gene segment, the D gene segment     being selected from a D in Table 13 found in said population and     with a cumulative frequency of at least 1 or 5%.     -   In another example there is provided     -   The antibody of clause 368, wherein the variable domain is         derived from the recombination of a human VH gene segment with a         human D gene segment and a human JH gene segment, the D gene         segment being selected from a D present in said population with         a cumulative frequency of at least 1 or 5%.     -   In an example, the D gene segment is found in the 1000 Genomes         database. In an example, the gene segment is found in Table 13. -   370. The antibody of clause 368 or 369, wherein the variable domain     is derived from the recombination of a human VH gene segment with a     human D gene segment and a human JH gene segment, the JH gene     segment being selected from a JH in Table 13 found in said     population and with a cumulative frequency of at least 1 or 5%.     -   In another example there is provided     -   The antibody of clause 368 or 369, wherein the variable domain         is derived from the recombination of a human VH gene segment         with a human D gene segment and a human JH gene segment, the JH         gene segment being selected from a JH present in said population         and with a cumulative frequency of at least 1 or 5%.     -   In an example, the JH gene segment is found in the 1000 Genomes         database. In an example, the gene segment is found in Table 13. -   371. An isolated VH domain identical to a variable domain as recited     in any one of clauses 368 to 370, optionally fused at its C-terminus     to a polypeptide (e.g., an antibody Fc). -   372. A pharmaceutical composition comprising the antibody or     variable domain of any one of clauses 368 to 371 together with a     pharmaceutically-acceptable excipient, diluent or a medicament     (e.g., a further antigen-specific variable domain, antibody chain or     antibody). -   373. An antibody heavy chain or VH domain (e.g., provided as part of     an antibody) for therapy and/or prophylaxis of a disease or medical     condition in a patient of European, East Asian, West African, South     Asian or Americas ancestry, wherein the heavy chain is a heavy chain     produced by the following steps (or is a copy of such a heavy     chain):—     -   (a) Selection of an antigen-specific antibody heavy chain or VH         domain from a non-human vertebrate (e.g., a mouse or a rat),         wherein the heavy chain or VH domain is derived from the         recombination of a human VH gene segment with a human D gene         segment and a human JH gene segment, the VH gene segment being         selected from a VH in Table 13 found in said population and with         a cumulative frequency of at least 1 or 5%;     -   (b) Optional humanization of the heavy chain by combining the         variable domain of the heavy chain with a human constant region;         or optional humanization of the selected VH domain by combining         with a human constant region.     -   In another embodiment, there is provided:—     -   An antibody heavy chain or VH domain (e.g., provided as part of         an antibody) for therapy and/or prophylaxis of a disease or         medical condition in a patient of European, East Asian, West         African, South Asian or Americas ancestry, wherein the heavy         chain is a heavy chain produced by the following steps (or is a         copy of such a heavy chain):—     -   (a) Selection of an antigen-specific antibody heavy chain or VH         domain from a non-human vertebrate (e.g., a mouse or a rat),         wherein the heavy chain or VH domain is derived from the         recombination of a human VH gene segment with a human D gene         segment and a human JH gene segment, the VH gene segment being         selected from a VH present in said population with a cumulative         frequency of at least 1 or 5%;     -   (b) Optional humanization of the heavy chain by combining the         variable domain of the heavy chain with a human constant region;         or optional humanization of the selected VH domain by combining         with a human constant region.     -   In an example, the VH gene segment is found in the 1000 Genomes         database. In an example, the gene segment is found in Table 13. -   374. The antibody heavy chain or VH domain of clause 373, wherein     the human constant region is as recited in clause 361 or 362. -   375. An antibody heavy chain or VH domain as recited in clause 373     or 374 for use in a medicament for therapy and/or prophylaxis of a     disease or medical condition in a patient of said ancestry. -   376. A method of treating and/or preventing a disease or medical     condition in a patient of European, East Asian, West African, South     Asian or Americas ancestry, the method comprising administering to     the patient a therapeutically or prophylactically-effective amount     of the antibody heavy chain or VH domain as recited in clause 373 or     374.     -   In embodiments herein, a Chinese patient can be a Han Chinese         patient.     -   In embodiments herein, a patient of European ancestry can be a         patient of Northern or Western European ancestry, Italian         ancestry, British or Scottish ancestry, Finnish ancestry or         Iberian ancestry.     -   In embodiments herein, a patient of East Asian ancestry can be a         patient of Han Chinese ancestry, Japanese ancestry Chinese Dai         ancestry, Vietnamese ancestry or Kinh ancestry.     -   In embodiments herein, a patient of West African ancestry can be         a patient of Yoruba ancestry, Luhya ancestry, Gambian ancestry         or Malawian ancestry.     -   In embodiments herein, a patient of Americas ancestry can be a         patient of African American ancestry, African Caribbean         ancestry, Mexican ancestry, Puerto Rican ancestry, Colombian         ancestry or Peruvian ancestry.     -   In embodiments herein, a patient of South Asian ancestry can be         a patient of Ahom ancestry, Kayadtha ancestry, Reddy ancestry,         Maratha ancestry, or Punjabi ancestry.     -   In an example of any aspect, the cumulative frequency is at         least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,         50, 55, 60, 65, 70, 75, 80, 85, 90 or 95%.     -   It will be understood that particular embodiments described         herein are shown by way of illustration and not as limitations         of the invention. The principal features of this invention can         be employed in various embodiments without departing from the         scope of the invention. Those skilled in the art will recognize,         or be able to ascertain using no more than routine study,         numerous equivalents to the specific procedures described         herein. Such equivalents are considered to be within the scope         of this invention and are covered by the claims. All         publications and patent applications mentioned in the         specification are indicative of the level of skill of those         skilled in the art to which this invention pertains. All         publications and patent applications are herein incorporated by         reference to the same extent as if each individual publication         or patent application was specifically and individually         indicated to be incorporated by reference. The use of the word         “a” or an when used in conjunction with the term “comprising” in         the claims and/or the specification may mean “one,” but it is         also consistent with the meaning of “one or more,” “at least         one,” and “one or more than one.” The use of the term or in the         claims is used to mean “and/or” unless explicitly indicated to         refer to alternatives only or the alternatives are mutually         exclusive, although the disclosure supports a definition that         refers to only alternatives and “and/or.” Throughout this         application, the term “about” is used to indicate that a value         includes the inherent variation of error for the device, the         method being employed to determine the value, or the variation         that exists among the study subjects.     -   As used in this specification and claim(s), the words         “comprising” (and any form of comprising, such as “comprise” and         “comprises”), “having” (and any form of having, such as “have”         and “has”), “including” (and any form of including, such as         “includes” and “include”) or “containing” (and any form of         containing, such as “contains” and “contain”) are inclusive or         open-ended and do not exclude additional, unrecited elements or         method steps     -   The term or combinations thereof as used herein refers to all         permutations and combinations of the listed items preceding the         term. For example, “A, B, C, or combinations thereof is intended         to include at least one of: A, B, C, AB, AC, BC, or ABC, and if         order is important in a particular context, also BA, CA, CB,         CBA, BCA, ACB, BAC, or CAB. Continuing with this example,         expressly included are combinations that contain repeats of one         or more item or term, such as BB, AAA, MB, BBC, AAABCCCC,         CBBAAA, CABABB, and so forth. The skilled artisan will         understand that typically there is no limit on the number of         items or terms in any combination, unless otherwise apparent         from the context.     -   Any part of this disclosure may be read in combination with any         other part of the disclosure, unless otherwise apparent from the         context.     -   All of the compositions and/or methods disclosed and claimed         herein can be made and executed without undue experimentation in         light of the present disclosure. While the compositions and         methods of this invention have been described in terms of         preferred embodiments, it will be apparent to those of skill in         the art that variations may be applied to the compositions         and/or     -   methods and in the steps or in the sequence of steps of the         method described herein without departing from the concept,         spirit and scope of the invention. All such similar substitutes         and modifications apparent to those skilled in the art are         deemed to be within the spirit, scope and concept of the         invention as defined by the appended claims.

The present invention is described in more detail in the following non limiting prophetic Examples.

EXAMPLES Example 1 Recombineered BAC Vectors to Add Polymorphic V-Regions to the Mouse Genome

FIG. 1 through 3 depict recombineering methods (see references above) that can be used to introduce polymorphic V-gene regions into genomic DNA. In one embodiment, a genomic fragment from the human heavy chain region is inserted into a bacterial artificial chromosome (BAC) vector by standard techniques. Preferably, such a BAC, which can range in size from 20-kb to 200-kb or more, can be isolated from libraries of BACs by standard techniques including sequence searches of commercially available libraries or by hybridization to bacterial colonies containing BACs to identify those with a BAC of interest.

A BAC is chosen that has several VH gene segments; in FIG. 1, these are generically identified as VH[a] through VH[z] for example. One skilled in the art will readily identify appropriate genomic fragments, for example, an approximately 120-kb fragment from human VH5-78 through VH1-68 which includes 5 endogenous active VH gene segments and 7 VH pseudogenes. Using recombineering techniques, the endogenous VH gene segments can be replaced by polymorphic VH or VL gene segments. In this example, two steps are required. The first step replaces the V-region coding exon of an endogenous VH gene segment with a positive-negative selection operon, in this example, an operon encoding an ampicillin resistance gene (Amp) and a streptomycin-sensitizing ribosomal protein (rpsL). Certain strains of bacteria can be selected for the absence of the rpsL gene by resistance to streptomycin. Short stretches of DNA homologous to sequences flanking the endogenous VH gene exon are placed 5′ and 3′ of the rpsL-Amp operon. In the presence of appropriate recombination factors per standard recombineering techniques (see references above) recombination between the operon fragment and the BAC will result in replacement of the endogenous VH gene exon with the operon (FIG. 1 a) which are selected by resistance to ampicillin. The second step uses the same homologous sequences in order to replace the inserted operon with a desired polymorphic VH gene segment. In this example, a human VH1-69 gene is inserted (FIGS. 1 b and 1 c). In particular the *02 variant of VH1-69 is used [ref IMGT and FIG. 5]. Successful integrations of the polymorphic VH gene segment are selected in bacteria that become resistant to streptomycin due to the loss of the operon, specifically the rpsL portion.

In this example, the two step process as described can be repeated for each of the endogenous VH gene segments or for as many endogenous gene segments that one wishes to replace with polymorphic V gene segments (FIG. 1 d).

As is apparent, any polymorphic V gene segment can be inserted in this manner and any endogenous V gene segment can act as a target, including pseudogenes. V gene segments in each of the heavy chain and two light chain loci can be replaced using this technique with appropriate genomic fragments available as BAC inserts.

FIG. 2 depicts another method for creating a genomic fragment encoding polymorphic V gene segments. In this example, polymorphic V gene segments are inserted into a region of genomic DNA devoid of other genes, control elements or other functions. Such ‘desert’ regions can be selected based on sequence analysis and corresponding DNA fragments cloned into BACs or identified in existing BAC libraries. Starting with such a genomic fragment, recombineering techniques can be used to insert polymorphic V gene segments at intervals of, for example, 10-kb. In this example, a 150-kb genomic fragment might accommodate insertion of up to 15 polymorphic V gene segments. Insertion of the segments is a two-step process. The first recombineering step inserts the rpsL-Amp operon at a specific site. Sequences homologous to a specific site are used to flank the operon. These are used by the recombineering system to insert the element specifically into the BAC genomic fragment and positive events are selected by resistance to ampicillin (FIG. 2 a). The second step replaces the operon in the genomic fragment with a polymorphic V gene segment by a similar recombineering step using the same sequence homology (FIG. 2 b). In this example, both

exons and promoter element of a polymorphic VH gene segment are inserted, resulting in replacement of the rpsL-Amp operon and therefore resistance to streptomycin (FIG. 2 c).

The two step technique for inserting polymorphic V gene segments into a specific site on the genomic fragment can be repeated multiple times resulting in a BAC genomic fragment with several polymorphic gene segments, including their promoter elements. It is apparent that the examples shown in FIGS. 1 and 2 can be combined wherein the technique for insertion can be used to add extra polymorphic V gene segments to a BAC genomic fragment as depicted in FIG. 1. One might choose to add these extra segments to an IG genomic fragment since such a fragment would be more amenable to proper IG gene expression once inserted into a non-human mammal's genome. It is known that a genomic fragment can have elements such as enhancers or elements that contribute to certain chromatin conformations, both important in wild-type gene expression.

FIG. 3 depicts an additional method to create genomic fragments with polymorphic V gene segments. This method depends upon the efficiency with which short (around 50 to 150 bases, preferably 100 bases) single stranded DNA fragments recombine with a homologous sequence using recombineering (Nat Rev Genet. 2001 October; 2(10):769-79; Recombineering: a powerful new tool for mouse functional genomics; Copeland N G, Jenkins N A, Court D L). The recombinases used in recombineering preferentially bind and use such short single-stranded fragments of DNA as a substrate for initiating homologous recombination. The efficiency can be as high as 10-2, that is, a positive event can be found in approximately 100 randomly picked (not selected) clones resulting from recombineering. A positive event in this example occurring when one or more single nucleotide changes introduced into the single-stranded fragment get transferred to the BAC insert containing V gene segments and surrounding genomic DNA, said nucleotide change or changes occurring at a homologous sequence on the BAC.

Polymorphic V gene segments can differ from endogenous V gene segments by only 1 or 2, or up to 10 or 15 nucleotide changes, for example. An example of such nucleotide polymorphisms are depicted in FIG. 5. Short single stranded regions that encompass the polymorphic nucleotide changes can be chemically synthesized using standard techniques. The resulting single stranded DNA fragments are introduced into bacteria and via recombineering techniques approximately 1 in 100 BAC fragments will have incorporated the polymorphic nucleotides via homologous incorporation of the single stranded fragment (FIG. 3 a). BACs with the desired nucleotide change can be identified by screening for example several hundred individual clones by polymerase chain reaction (PCR) amplification and sequencing, both by standard techniques. In the example, two nucleotide changes will convert a VH1-69*01 gene segment into a VH1-69*02 gene segment (FIG. 3 b).

It is clear that this process can be repeated for multiple endogenous V gene segments contained on a single BAC genomic fragment. In addition, the techniques depicted in FIG. 2 can be used to add additional polymorphic V gene segments by insertion into regions between existing V gene segments. As would be evident to one skilled in the art, a combination of these techniques can be used to create numerous variations of both polymorphic and endogenous human V gene segments. And it would be evident that several different genomic fragments with engineered polymorphic V gene segments and endogenous human V gene segments can be combined to create even more variations.

Example 2 Adding Polymorphic V-Regions to the Genome Using SRMCE of Modified BACs

Modified BACs with polymorphic V gene segments created using the methods described in Example 1 can be used to alter the genome of non-human mammals. These alterations can result in an intact IG locus in which normal immunoglobin region recombination results in VDJ or VJ combinations which includes the human V gene segments. An example of how such an animal can be created is by altering the genome of, for example, mouse embryonic stem (ES) cells using the strategy outlined in FIG. 4.

One technique to integrate modified BACs with polymorphic V gene segments into a genome is sequential recombinase mediated cassette exchange (SRMCE). The technique is described in WO2011004192 (Genome Research Limited), which is incorporated here in its entirety by reference.

SRMCE provides for a locus modified with a ‘landing pad’ inserted at a specific location. This insertion can either be de novo via homologous recombination or as a consequence of a previous BAC insertion. In this example, the landing pad is inserted in the mouse IGH locus between the most 3′ J gene segment and the CA gene segment and a previous BAC insertion via SRMCE techniques have resulted in the addition of 5 human V gene segments and 2 V region pseudogenes. The landing pad has elements as shown in FIG. 4 that will allow the selection of correct insertion of a second targeting BAC fragment. The specificity of this insertion is provided by cre recombinase-mediated exchange between permissive lox sites. A lox site is permissive for recombination only with a compatible lox site. In this example, the loxP site will only recombine with loxP and lox2272 will only recombine with lox2272. This provides directionality to the insertion of the BAC fragment as depicted in FIGS. 4 b and 4 c.

ES cell clones with correct insertions are selected from a pool of clones without insertions or with non-productive insertions by resistance to puromycin. Resistance to puromycin results from the juxtaposition of an active promoter element, PGK, with the puroTK coding region. Correct insertions are verified by standard techniques including PCR of junctions, PCR of internal elements, Southern blotting, comparative genomic hybridization (CGH), sequencing and etc. In the example, correct lox2272-lox2272 and loxP-loxP recombination also results in two intact sets of piggyBac elements that did not exist prior to insertion. An intact piggyBac element is comprised of a set of inverted repeats which are depicted in the figure by “PB5′” and “PB3′”. An appropriated oriented set of piggyBac elements are the substrate of piggyBac transposase which can catalyse recombination between the elements, resulting in deletion of intervening sequences as well as both elements. The DNA remaining after a piggyBac transposition is left intact and is lacking any remnant of the piggyBac element. In the example, ES cell clones with successful piggyBac transposition are selected by loss of the active puroTK element which renders the cells resistant to the drug FIAU (FIGS. 4 c and 4 d).

The final product of the SRMCE method in this example is a IGH locus with several polymorphic V gene segments inserted along with a set of endogenous unmodified VH gene segments between sequences of the mouse genome on the 5′ side and the mouse IGH constant region gene segments on the 3′ side. The polymorphic V gene segments are positioned such that they can participate in the recombination events associated with B cell maturation yielding VDJ gene segments. These gene

segments can then be transcribed and spliced to the mouse constant region. Translation of these transcripts will result in the production of an antibody heavy chain encoded by the polymorphic V gene segment, a human DH gene segment, a human JH gene segment and a mouse constant heavy chain gene segment.

As is well known to those skilled in the art, an ES cell clone can be used to create a line of genetically modified mice via injection of said cells into a mouse blastocyst embryo, transferring the injected embryo to a suitable recipient and breeding the chimeric offspring that result. The modified gene locus can be propagated through breeding and made either heterozygous or homozygous depending on the genetic cross.

It is evident from the structure of the IGH locus provided in this example and by knowledge of the mechanisms involved in B cell receptor (BCR) and antibody gene rearrangements that a large set of different combinations of polymorphic V gene segments with various DH and JH gene segments will result and these can contribute to a large repertoire of functional antibody genes in a population of B cells in genetically modified animals. In this example, several different human VH1-69 polymorphs are incorporated to provide superhuman VH diversity. This particular VH gene segment is known to be prevalent in antibodies that bind infectious disease pathogens (such as influenza virus) and therefore the antibody repertoire of a mouse with the genetic modification of this example would be expected to produce antibodies with a bias in favour of those that bind infectious disease pathogens. The repertoire, in other words, would have a larger subset of antibodies with superior affinities for pathogen antigens. Examples of such pathogens include influenza virus, hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1) (see also table above).

Example 3 Alignment of 13 VH1-69 Alleles

Building a more diverse antibody repertoire by incorporating additional V gene segment polymorphs requires availability of polymorphic variants of V gene segments. One source of such variants include sequence databases. In this example, 13 distinct variants of the VH1-69 gene segment are provided.

These variant sequences and comparisons are drawn from the “IMmunoGeneTics” IMGT Information System (www.imgt.com) database. FIG. 5 is a diagram of the alignment of variants *02 through *13 with the *01 variant. The VH1-69*01 nucleotide and amino acid sequence is provided at the top of the figure. Where the remaining variants are identical to the *01 variant sequence a dash is inserted below the sequence. Nucleotide differences are noted alongside the appropriate variant and if the sequence change results in a protein coding change, the amino acid change is indicated above the triplet.

FIG. 5 depicts between 1 and 4 amino acid changes for each variant in comparison to the *01 variant. All of the amino acid changes occur in the part of the heavy chain protein encoding the complementarity determining regions (CDRs). These regions are responsible for antigen specificity and the affinity of the antibody for the antigen. It is evident that providing additional polymorphic CDRs in a repertoire of antibodies will increase the likelihood of there being an antibody with superior binding characteristics for various antigens. In several reports, it has been observed that the VH1-69-encoded variable region of the heavy chain is often found in antibodies that bind influenza virus, HCV and HIV-1 antigens (see table above). Therefore incorporating the polymorphic V gene segments of this example into a transgenic animal model using the methods of Examples 1 and 2 would likely result in an antibody repertoire in said transgenic animal with more antibodies that bind to antigens associated with these and other pathogens. And as is known in the art, a larger repertoire increases the probability of finding monoclonal antibodies using, for example, hybridoma technology, that bind with high affinity and specificity to a desired antigen.

This disclosure therefore describes in these examples a transgenic mouse model which can be immunized with pathogen or other antigens. Plasma B cells from such an immunized mouse can be used to make a hybridoma library that can be screened for production of antibodies that bind the pathogen antigens. This library will be superior to libraries from traditional transgenic mice for finding such antibodies given the addition of polymorphic VH1-69 gene segments to the IGH locus in said transgenic mouse.

These examples are not limiting to the human polymorphic V gene segments that can be chosen or to the methods used to introduce them into an animal model. The method can be used to construct a transgenic locus with immunoglobulin D and/or J segments. The V, D, J segments can be from a plurality of human sources (optionally more than one human ethnic population).

Example 4 Human IgH JH Gene Variants Selected from the 1000 Genomes Database

Data is presented for human JH2, 5 and 6 variants. In Tables 10A, 11A and 12A samples from humans from various populations are listed where the sequence analysis of the inventors has revealed the presence of polymorphisms in one or both IgH JH alleles. The population codes are explained in Table 8 above. The polymorphisms are nucleotide variants from JH2, 5 and 6 reference sequences (SEQ ID NOs: 1, 2 and 3 respectively; see below). All references are sequences taken from the Ensembl database (www.ensembl.org). The JH5 reference is human IgH J5-001 disclosed in that database. The JH6 reference is human IgH J6-001 disclosed in that database. The JH2 reference is human IgH J2-001 disclosed in that database.

The reference nucleotide and encoded amino acid sequences are shown on the next page. Alignments with encoded amino acid sequences are also provided, including the corresponding position numbers on human chromosome 14.

Variant Frequencies are shown in Tables 10A, 11A and 12A and these relate to the frequency of the variants in the 1000 Genomes Database (release current at October 2011).

Tables 10B, 11B and 12B show the non-synonymous nucleotide polymorphisms in the human JH variants, as sorted by the present inventors from the 1000 Genomes database. Position numbers corresponding to nucleotide positions on human chromosome 14 are shown for variant positions (chromosome 14 being the chromosome bearing the IgH locus in humans). Thus, for example, the first entry in Table 11B is “14:106330027:A/C” which refers to a position in a variant JH5 sequence wherein the position corresponds to position 106,330,027 on human chromosome 14, such position being A (adenine) in the reference sequence. The “C” indicates that the present inventors observed a mutation to cytosine at this position in the variants found in the 1000 Genomes database. This change leads to a change at the amino acid level of the encoded sequence (i.e., a “non-synonymous” change), in this case a change from a serine (found in the reference) to an alanine in the variant.

Example 5 Human Antibody Gene Segment Variant Identification & Population Analysis

The genomic coding region coordinates for each target gene for variant analysis were identified from the Ensembl WWW site (www.ensembl.org) using coordinates from the GRCh.p8 Human Genome assembly (www.ncbi.nlm.nih.gov/projects/genome/assembly/grc). Using the collected gene location coordinates, variant data was extracted from the public ftp site of the 1000 Genomes Project using the Perl ‘Variant Pattern Finder’ (VPF—www.1000genomes.org/variation-pattern-finder-api-documentation).

Data extracted by VPF was post processed using software to extract all non-synonymous (NSS) variants with their associated genotype calls. Genotypes calls were assembled to form unique haplotypes, representing groups of NSS variants associated with 1000 Genome population groups and frequency of occurrence within those populations.

The output of the analysis results in tables such as in Table 13. The main body of the table describes each haplotype in turn giving a unique ID for that gene (in the range a-z,aa-zz), the population frequencies and occurrence in individuals and unique population groups; one or more subsequent columns describe the DNA base calls at each location that form the haplotype giving both the base from the reference sequence or the variant base call.

-   Table 13 was constructed in this manner. The table can be read as     follows:

The first four columns (left to right) consist of (1) the haplotype ID letter ('ref indicates reference—the DNA base call at each genomic location from the GRCh37 Human Reference Assembly) (2) the observed cumulative frequency of the haplotype among the different populations (3) the number of individuals in which a specific haplotype was observed (4) the number of unique population groups that the identified individuals belong to (the actual population group identifiers are displayed as a string of ID's in the most right hand column for each haplotype. For example haplotype ‘a’ has a population ID string of ‘3,4,9,13’).

The populations are numbered as follows (population labels being according to 1000 Genomes Project nomenclature)

1=ASW; 2=CEU; 3=CHB; 4=CHS; 5=CLM; 6=FIN; 7=GBR; 8=IBS; 9=JPT; 10=LWK; 11=MXL; 12=PUR; 13=TSI; 14=YRI.

Subsequent columns detail a single point variant and have the following format (top to bottom) (1) the human genomic location of the variant (format [chromosome number]: [location] e.g. ‘14:106204113’); (2) The identifier for the point variant as defined in DbSNP (www.ncbi.nlm.nih.gov/projects/SNP/); (3) One or additional rows show the amino acid change as result of the variant for a specific transcript (denoted by the Ensembl transcript ID in the most right-hand column for each row), the format is the amino acid in the reference sequence followed by ‘->’ and the amino acid caused by the substitution of the variant in the reference sequence (e.g. ‘Gly->Arg’ means a that the translated reference sequence would result in a glycine at that location, whereas the substitution of the identified variant would result in translated protein containing arginine) using the IUPAC three letter amino acid codes (http://pac.iupac.org/publications/pac/pdf/1972/pdf/3104×0639.pdf). Subsequent rows (one per haplotype) show the DNA base at each location, bases matching the reference sequence are shown in black on white back ground, bases varying from the reference are shown as white text on a black background.

The most right-hand column contains the Ensembl transcript ID's (e.g. ‘ENST00000390542’) for each of the gene transcript and relates to the amino acid changes to the left of this column.

Because the transcripts are differing lengths each variant position may or may not have an associated amino acid change at the that position.

Example 6 Transgenic Mice. B-cells. Hebridomas. Antibodies & Heavy Chains Based on Human JH6*02

A functional human gene segment repertoire (from VH2-26 to JH6, see the IMGT database for the structure of the human IgH locus;

http://www.imgt.org/IMGTrepertoire/index.php?section=LocusGenes&repertoire=locus&species=human&group=IGK) was sectored by the inventors to produce two different transgenic heavy chain alleles (denoted S2F and S3F) and corresponding mice. The transgenic alleles were expressed in the mice and the heavy chain repertoires were assessed at the RNA transcript level. Deep sequence analysis was carried out using Bioinformatics methods to assess V, D and JH gene usage, including in variable domain sequences having a HCDR3 length of at least 20 amino acids. Endogenous, mouse variable region gene segments were inactivated by inversion (as per the method described in WO2011004192, this disclosure being incorporated herein by reference).

Sequencing of Human Donor DNA Samples: Identification of Conserved JH6*02 Variant

DNA samples from 9 anonymised consenting human donors were obtained by taking cheek swabs.

The samples were processed and the DNA Samples were extracted follow the protocol of QIAamp DNA Mini Kit (Cat. No. 51304, Qiagen).

PCR reactions were set up to amplify the JH6 region and PCR products were sequenced (PCR Oligos sequence: Fwd. 5′-AGGCCAGCAGAGGGTTCCATG-3′ (SEQ ID NO: 444), Rev. 5′-GGCTCCCAGATCCTCAACCCAC-3′ (SEC) ID NO: 445)).

Sequence analysis was carried out by comparing to the JH6 reference sequence from IMGT annotated database (http://www.imgt.org/) and this identified that all 9 donor genomes contained the human JH6*02 variant, with this variant being in the homozygous state in 7 out of the 9 donors. The inventors also consulted the genomic sequences publicly available for Jim Watson and Craig Venter at Ensembl human genome database [http://www.ensembl.org/]. These too contained the human JH6*02 variant. This confirmed to the inventors that human JH6*02 is a common, conserved variant in humans, and thus a good candidate for construction of a transgenic IgH locus as per the invention

Identification of Suitable Human DNA Sequence BACs

A series of human bacterial artificial chromosome (BAC) clones were identified from Ensemble (http://www.ensembl.org/index.html) or UCSC (http://genome.ucsc.edu/) human database searches based on gene name (IGH) or location (chromosome 14: 106026574-107346185). Seven human RP11 BAC clones (see an extract of the UCSC database in FIG. 10, identified BACs being circled) were selected, RP11-1065N8 BAC carrying human JH6*02. In total, the following BACs were identified as sources of human IgH locus DNA: RP11-1065N8, RP11-659B19, RP11-14117, RP-112H5, RP11-101G24, RP11-12F16 and RP11-47P23.

With a similar approach, different BAC clones (e.g., different RP11 clone IDs or different sources from RP11) or genetically engineered BACs can be selected for insertion into the mouse IGH locus to provide different sets of human repertoires in the transgenic mouse.

Construction of Transgenic IgH Loci

Insertion of human heavy gene segments from a 1st IGH BAC (RP11-1065N8) into the IGH locus of mouse AB2.1 ES cells (Baylor College of Medicine) was performed to create a heavy chain allele denoted the S1 allele. The inserted human sequence corresponds to the sequence of human chromosome 14 from position 106494908 to position 106328951 and comprises functional heavy gene segments VH2-5, VH7-4-1, VH4-4, VH1-3, VH1-2, VH6-1, D1-1, D2-2, D3-9, D3-10, D4-11, D5-12, D6-13, D1-14, D2-15, D3-16, D4-17, D5-18, D6-19, D1-20, D2-21, D3-22, D4-23, D5-24, D6-25, D1-26, D7-27, JH1, JH2, JH3, JH4, JH5 and JH6 (in 5′ to 3′ order), wherein the JH6 was chosen to be the human JH6*02 variant. The insertion was made between positions 114666435 and 114666436 on mouse chromosome 12, which is upstream of the mouse Ĉ region. The mouse VH, D and J H gene segments were retained in the locus, immediately upstream of (5′ of) the inserted human heavy chain DNA.

A second allele, S2 was constructed in which more human functional VH gene segments were inserted upstream (5′) of the 5′-most VH inserted in the 51 allele by the sequential insertion of human DNA from a second BAC (BAC2). The inserted human sequence from BAC2 corresponds to the sequence of human chromosome 14 from position 106601551 to position 106494909 and comprises functional heavy chain gene segments VH3-13, VH3-11, VH3-9, VH1-8, VH3-7. The mouse VH, D and JH gene segments were retained in the locus, immediately upstream of (5′ of) the inserted human heavy chain DNA. In a subsequent step, these were inverted to inactivate them, thereby producing S2F mice in which only the human heavy chain variable region gene segments are active.

A third allele, S3 was constructed in which more human functional VH gene segments were inserted upstream (5′) of the 5′-most VH inserted in the S2 allele by the sequential insertion of human DNA from a third BAC (BAC3). The inserted sequence corresponds to the sequence of human chromosome 14 from position 106759988 to position 106609301, and comprises functional heavy chain gene segments, VH2-26, VH1-24, VH3-23, VH3-21, VH3-20, VH1-18, and VH3-15. The mouse VH, D and JH gene segments were retained in the locus, immediately upstream of (5′ of) the inserted human heavy chain DNA. In a subsequent step, these were inverted to inactivate them, thereby producing S3F mice in which only the human heavy chain variable region gene segments are active.

Mice bearing either the S2F or S3F insertion into an endogenous heavy chain locus were generated from the ES cells using standard procedures. The other endogenous heavy chain locus was inactivated in the mice by insertion of an inactivating sequence comprising neoR into the mouse JH-Ĉ intron (to produce the “HA” allele).

Immunisation Procedure

Transgenic mice of the S2F or S3F genotype were primed with 20-40 ug recombinant proteins obtained commercially or produced in house with Antigen 1 (OVA (Sigma A7641); Antigen 2 (a human infectious disease pathogen antigen) and Antigen 3 (a human antigen) via the ip route in complete Freunds adjuvant (Sigma F 5881) and 10 ug/animal CpG (CpG oligo; Invivogen, San Diego, Calif., USA) and then boosted twice in about two weekly intervals with about half the amount of antigen in incomplete Freunds adjuvant (Sigma F 5506) and 10 ug/animal CpG. Final boosts were administered two weeks later iv without any adjuvant and contained 5-10 ug protein in PBS.

Hybridoma Fusion Procedure

Spleens were taken 3 days after the final boost and spleenocytes were treated with CpG (25 ̂m final concentration) for and left until the following day. Cells were then fused with SPO/2 Ag14 myeloma cells (HPA Cultures Cat No 85072401) using a BTX ECM2001 electrofusion instrument. Fused cells were left to recover for 20 minutes then seeded in a T75 flask until next morning. Then the cells were spun down and plated out by dilution series on 96-well culture plates and left for about 10 days before screening. Media was changed 1-3 times during this period.

Screening

Culture supernatants of the hybridoma wells above were screened using homogenious time resolved fluorescence assay (htrf) using Europium cryptate labelled anti-mouse IgG (Cisbio anti-mouse Ig Europium Cryptate) and a biotin tagged target antigen with a commercially available streptavidin conjucated donor (Cisbio; streptaviding conjugated D2) or by IgG-specific 384 well ELISA. Positive wells identified by htrf were scaled to 24-well plates or immediately counterscreened using an IgG-specific detection ELISA method. Positives identified by primary ELISA screen were immediately expanded to 24-well plates. Once cultures were expanded to 24-well stage and reached conflueny, supernatants were re-tested using htrf or IgG-specific ELISA to confirm binding to target antigen. Supernatant of such confirmed cultures were then also analysed by surface plasmon resonance using a BioRad ProteOn XPR36 instrument. For this, antibody expressed in the hybridoma cultures was captured on a biosensor GLM chip (BioRad 176-512) which had an anti-mouse IgG (GE Healthcare BR-1008-38)) covalently coupled the biosensor chip surface. The antigen was then used as the analyte and passed over the captured hybridoma antibody surface. For Antigen 2 and Antigen 3, concentrations of 256 nM, 64 nM, 16 nM, 4 nM and 1 nM were typically used, for Antigen 1, concentrations of 1028 nM, 256 nM, 64 nM, 16 nM and 4 nM were typically used, binding curves were double referenced using a 0 nM injection (i.e. buffer alone). Kinetics and overall affinities were determined using the 1:1 model inherent to the BioRad ProteOn XPR36 analysis software.

Any clones with confirmed binding activity were used for preparing total RNA and followed by PCR to recover the heavy chain variable region sequences. Standard 5′-RACE was carried out to analyse RNA transcripts from the transgenic heavy chain loci in the S2F and S3F mice. Additionally, deep sequence analysis of almost 2000 sequences produced by the mice was carried out.

Bioinformatics Analysis

Sequences for analysis were obtained from two different methods:

-   -   The first is from RNA extracted from the spleen: first cDNA         strand was synthesized using an oligo based on the Cmu region of         the mouse IGH locus as a PCR template. PCR was performed using         this oligo with an oligo dT-anchor primer. Then PCR product was         cloned into pDrive vector (Qiagen) and then sequenced.     -   The second is from hybridomas generated through electro-fusion:         total RNA was extracted from hybridoma lines of interest using         standard Trizol methods and frozen at −80 oC for long term         storage. cDNA was generated from 100 ng total RNA using standard         Superscript III reverse transcriptase and a gene-specific         reverse primer binding to all mouse IgG isotypes for heavy chain         and a mouse kappa constant region primer for the light chain         amplification. 2-3 ul of cDNA were then used as template in a         PCR reaction using Pfu DNA polymerase and a panel of degenerate         forward primers annealing to the leader sequence of the human         immunoglobulin variable domain as well as one mouse pan-IgG         reverse primer. PCR products were run out of a 1% agarose gel         and bands of approximately 350-450 basepairs extracted and         purified. DNA was then sequenced.

The sequences from the first method can either be from IgM from Naive mice or IgG from immunised mice. The samples from the second method are all from IgG from immunised mice, and specific to the immunizing antigen. Almost 2000 sequences were analysed.

The sequences were obtained as a pair of forward and reverse reads. These were first trimmed to remove low-quality base calls from the ends of the reads (trimmed from both ends until a 19 nucleotide window had an average quality score of 25 or more). The reads were combined together by taking the reverse complement of the reverse read, and aligning it against the forward read. The alignment scoring was 5 for a match, −4 for a mismatch, a gap open penalty of 10 and a gap extension penalty of 1. A consensus sequence was then produced by stepping through the alignment and comparing bases. When there was a disagreement the base with the highest quality value from sequencing was used.

The BLAST© (Basic Local Alignment Search Tool) (Camacho C., Coulouris G., Avagyan V., Ma N., Papadopoulos J., Beeler K., & Madden T. L. (2008) “BLAST©: architecture and applications.”BMC Bioinformatics 10:421 http://www.ncbi.nlm.nih.gov/pubmed/20003500) program ‘blastn’ was then used to find the germline J and V segments used in each sequence. A wordsize of 30 was used for V matching, and 15 for J matching. The database searched against was constructed from the NGS sequencing of the BACs which were used to generate the Kymouse.

If a sequence matched both a V and a J segment, the sequence between the two was then compared to a database of germline D segments in the mouse using ‘blastn’ with a wordsize of 4 and the options ‘blastn-short’ and ‘ungapped’. This was used to assign a D segment, if possible. The CDR3 was identified by searching for the conserved “TATTACTGT” sequence in the V segment, and the “CTGGGG” in the J segment. If these motifs were not found, then up to 4 mismatches were allowed. The IMGT definition of CDR3 was used, so the CDR3 length is calculated from after the “TGT” in the V to before the “TGG” in the J. Sequences with an out of frame junction (those which do not have a CDR3 nucleotide length divisible by 3) or which contained a stop codon (“TAA”, “TAG” or “TGA”) were excluded.

The identity of the matching V, J and D segments as well as the CDR3 length from this assignment were then saved as a table for downstream analysis. The ratio of IGHJ6*02 used increased from the naive to immunised mice, as well as being enriched in the sub-population of sequences with a long HCDR3 (defined as consisting of 20 or more amino acids):

All HCDR3 > 20 Total Total JH6 * 02% Count JH6 * 02% Count % HCDR3 > 20 Naive 22.31% 1340 91.11% 45 3.36% Immunised 37.50% 256 66.67% 9 3.52% Hybridoma 36.13% 119 63.64% 11 9.24%

This shows that the JH6*02 gene segment is selected for by immunisation, as the proportion of JH6*02 usage increases after immunisation. JH6*02 is also used in the majority of antibodies with a long HCDR3 length, which is desirable for targets which are specifically bound by long HCDR3 length antibodies.

SEQ ID NO: 1 (JH5 Reference) T T G A C C A A G C T G G G G A C C C C G G T C C C T T G G G A C C A G T G G C A G A G G A G T C JH5 Alignment: (top line = SEQ ID NO: 1, Middle line = SEQ ID NO: 5, Bottom line = SEQ ID NO: 6) LgH J5001 106, 330, 072 106, 330, 071 106, 330, 068,  106, 106, 106, 106, 106, 106, 106, 106, 330, 041 106, 330, 082 106, 330, 027, 106, 330, 024 330, 067 330, 065 330, 065 330, 063 330, 062 330, 045, 330, 044 T T G A C C A A G C T G G G G A C C C C G C C T T G G G A C C A G T G G C A G A G G- A A C T G G T T C G A C C C C T G G C T G G — C A C C G T C C T C A G W P W G Q G T L V T V S S SEQ ID NO: 2(JH6Reference) A T G A T G A T G A T G A T G A T G T A C C T G C A G A C C C C G T T T C C C T G G T G C C A G T G G C A G A G G A G T JH6 Alignment: (top line = SEQ ID NO: 2, Middle line = SEQ ID NO: 7, Bottom line = SEQ ID NO: 8) LgH J6001 106, 329, 468 106, 329, 453 106, 329, 452, 106, 329, 451 106, 329, 435 T T G A C C A A G C T G G G G A C C C C G C C T T G G G A C C A G T G G C A G A G G- A A 106, 329, 426 106, 106, 106, 106, 106, 106, 329, 413 329, 411 329, 408 329, 419 329 417 329, 414 C C C T G G T G C C A G T G G C A G A G G A G T C G G G A C C A C G G T C A C C G T C T C C T C A G G T T V T V S S SEQ ID NO: 3 JH 2 Reference) A T G A C C A T G A A G C T A G A G A C C C C G G C A C C G T G G G A C C A G T G A C A G A G G A G T C JH2 Alignment: (top line = SEQ ID NO: 3, Middle line = SEQ ID NO: 9, Bottom line = SEQ ID NO: 10) 106, 106, 106, IgH J5-001 331, 460 331, 455 331, 453 106, 331, 453 A T G A C C A T G A A G C T A G A G A C C C C G G C A C C G T G G G A C C A G T G A C A G A G G A G T C T A C T G G — A C T T C G A — C T C T G G G G C C G T G G C A C C C T G G T C A C T G T C T C C T C A G Y W Y F D L W G R G T L V T V S S

TABLES

In the tables, the notation is illustrated by the following example

IGLV1 40 G1 40 * 02 X53936 :g9 > Ic1) > g, L4 > VI

Polymorphic variant IGV lambda VI-40*02 has Genbank Accession No. X53936 and when compared to the *01 variant, the VI-40*02 variant has mutations at positions 9, 10 and 4. For example, at position 9, a “C” appears instead of a “G” that is present in the *01 variant. The “|” is simply a notation separator, and does not indicate any mutation. For example the “g282|” notation indicates no change (i.e., position 282 is a g). “del#” means that the residue at that position is absent.

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REFERENCES

-   1. Nat. Biotechnol. 2005 September; 23(9):1117-25; Human antibodies     from transgenic animals; Lonberg N. -   2. J Clin Invest. 1992 March; 89(3):729-38; Immunoglobulin light     chain variable region gene sequences for human antibodies to     Haemophilus influenzae type b capsular polysaccharide are dominated     by a limited number of V kappa and V lambda segments and VJ     combinations; Adderson E E, Shackelford P G, Insel R A, Quinn A,     Wilson P M, Carroll W L. -   3. J. Immunol. 1993 Oct. 15; 151(8):4352-61; Clonal characterization     of the human IgG antibody repertoire to Haemophilus influenzae type     b polysaccharide. V. In vivo expression of individual antibody     clones is dependent on Ig CH haplotypes and the categories of     antigen; Chung G H, Scott M G, Kim K H, Kearney J, Siber G R,     Ambrosino D M, Nahm M H. -   4. J. Immunol. 1998 Dec. 1; 161(11):6068-73; Decreased frequency of     rearrangement due to the synergistic effect of nucleotide changes in     the heptamer and nonamer of the recombination signal sequence of the     V kappa gene A2b, which is associated with increased susceptibility     of Navajos to Haemophilus influenzae type b disease; Nadel B, Tang     A, Lugo G, Love V, Escuro G, Feeney A J. -   5. J Clin Invest. 1996 May 15; 97(10):2277-82; A defective Vkappa A2     allele in Navajos which may play a role in increased susceptibility     to Haemophilus influenzae type b disease; Feeney A J, Atkinson M J,     Cowan M J, Escuro G, Lugo G. -   6. Infect Immun. 1994 September; 62(9):3873-80; Variable region     sequences of a protective human monoclonal antibody specific for the     Haemophilus influenzae type b capsular polysaccharide; Lucas A H,     Larrick J W, Reason D C. -   7. J Clin Invest. 1993 June; 91(6):2734-43; Restricted     immunoglobulin VH usage and VDJ combinations in the human response     to Haemophilus influenzae type b capsular polysaccharide. Nucleotide     sequences of monospecific anti-Haemophilus antibodies and     polyspecific antibodies cross-reacting with self antigens; Adderson     E E, Shackelford P G, Quinn A, Wilson P M, Cunningham M W, Insel R     A, Carroll W L. -   8. J Clin Invest. 1993 March; 91(3):788-96; Variable region     expression in the antibody responses of infants vaccinated with     Haemophilus influenzae type b polysaccharide-protein conjugates.     Description of a new lambda light chain-associated idiotype and the     relation between idiotype expression, avidity, and vaccine     formulation. The Collaborative Vaccine Study Group; Granoff D M,     Shackelford P G, Holmes S J, Lucas A H. -   9. Infect Immun. 1994 May; 62(5):1776-86; Variable region sequences     and idiotypic expression of a protective human immunoglobulin M     antibody to capsular polysaccharides of Neisseria meningitidis group     B and Escherichia coli K1; Azmi F H, Lucas A H, Raff H V, Granoff D     M. -   10. J Clin Invest. 1992 December; 90(6):2197-208; Sequence analyses     of three immunoglobulin G anti-virus antibodies reveal their     utilization of autoantibody-related immunoglobulin Vh genes, but not     V lambda genes; Huang D F, Olee T, Masuho Y, Matsumoto Y, Carson D     A, Chen P P. -   11. Science. 2011 Aug. 12; 333(6044):834-5, Biochemistry. Catching a     moving target, Wang T T, Palese P -   12. Science. 2009 Apr. 10; 324(5924):246-51. Epub 2009 Feb. 26;     Antibody recognition of a highly conserved influenza virus epitope;     Ekiert D C, Bhabha G, Elsliger M A, Friesen R H, Jongeneelen M,     Throsby M, Goudsmit J, Wilson I A. -   13. PLoS One. 2008; 3(12):e3942. Epub 2008 Dec. 16; Heterosubtypic     neutralizing monoclonal antibodies cross-protective against H5N1 and     H1N1 recovered from human IgM© memory B cells; Throsby M, van den     Brink E, Jongeneelen M, Poon L L, Alard P, Cornelissen L, Bakker A,     Cox F, van Deventer E, Guan Y, Cinatl J, ter Meulen J, Lasters I,     Carsetti R, Peiris M, de Kruif J, Goudsmit J. -   14. Nat Struct Mol Biol. 2009 March; 16(3):265-73. Epub 2009 Feb.     22,Structural and functional bases for broad-spectrum neutralization     of avian and human influenza A viruses, Sui J, Hwang W C, Perez S,     Wei G, Aird D, Chen L M, Santelli E, Stec B, Cadwell G, Ali M, Wan     H, Murakami A, Yammanuru A, Han T, Cox N J, Bankston L A, Donis R O,     Liddington R C, Marasco W A. -   15. Science. 2011 Aug. 12; 333(6044):843-50. Epub 2011 Jul. 7, A     highly conserved neutralizing epitope on group 2 influenza A     viruses, Ekiert D C, Friesen R H, Bhabha G, Kwaks T, Jongeneelen M,     Yu W, Ophorst C, Cox F, Korse H J, Brandenburg B, Vogels R,     Brakenhoff J P, Kompier R, Koldijk M H, Cornelissen L A, Poon L L,     Peiris M, Koudstaal W, Wilson I A, Goudsmit J.

LENGTHY TABLES The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140041067A1). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3). 

1. A non-human vertebrate whose genome comprises an immunoglobulin heavy chain locus comprising one or more VH gene segments, one or more D gene segments, and one or more J segments comprising human gene segment JH6*02, upstream of a constant region; wherein said gene segments in said heavy chain locus are operably linked to said constant region, and said vertebrate is functional to recombine a said VH gene segment, a said D gene segment and said human JH6*02 to form a VH-D-JH6*02 variable region gene.
 2. The vertebrate of claim 1, wherein said vertebrate has been immunised with a target antigen and comprises an antibody heavy chain comprising a variable domain encoded by said VH-D-JH6*02 gene produced by recombination of said heavy chain gene segments with said human JH6*02, wherein said variable domain comprises an HCDR3 having a length of at least 20 amino acids.
 3. A non-human vertebrate cell whose genome comprises an immunoglobulin heavy chain locus comprising one or more VH gene segments, one or more D gene segments and one or more J segments comprising human gene segment JH6*02, upstream of a constant region, wherein said gene segments of said heavy chain locus are operably linked to said constant region and said vertebrate is functional to recombine a said VH segment, a said D segment and said human JH6*02 gene segment to form a VH-D-JH6*02 variable region gene.
 4. The cell of claim 3, which is an ES cell, wherein said ES cell is capable of developing into a mouse comprising said immunoglobulin heavy chain locus in its germ line, and wherein said mouse comprises an antibody-producing cell that expresses an antibody comprising said heavy chain.
 5. The vertebrate of claim 1, wherein said heavy chain locus comprises a human JH6*02 recombination signal sequence (RSS) positioned upstream of and operably linked to said JH6*02 gene segment.
 6. The cell of claim 3, wherein the heavy chain locus comprises a human JH6*02 recombination signal sequence (RSS) positioned upstream of and operably linked to said JH6*02 gene segment.
 7. The vertebrate of claim 5, wherein said RSS is SEQ ID NO: 238 or, wherein said SEQ ID NO: 238 comprises a flanking 9mer, a central 22mer and a flanking 7mer, and said sequence comprises identical 9mer and 7mer flanking sequences and said central sequence is at least 70% identical to said 22mer central sequence of SEQ ID NO:
 238. 8. The cell of claim of claim 6, wherein the RSS is SEQ ID NO: 238 or, wherein said SEQ ID NO: 238 comprises a flanking 9mer, a central 22mer and a flanking 7mer, and said sequence comprises identical 9mer and 7mer flanking sequences and said central sequence is at least 70% identical to said 22mer central sequence of SEQ ID NO:
 238. 9. The vertebrate of claim 6, wherein said RSS and JH6*02 are provided as SEQ ID NO:
 237. 10. The cell of claim 8, wherein said RSS and JH6*02 are provided as SEQ ID NO:
 237. 11. The vertebrate of claim 1, wherein said JH6*02 is the only JH6 gene segment in the genome.
 12. The cell of claim 3, wherein said JH6*02 is the only JH6 gene segment in said genome.
 13. The vertebrate of claim 1, wherein said J gene segments comprise a plurality of J gene segments comprising a 3′ J gene segment positioned downstream in said plurality and upstream of said constant region in said locus, and said 3′ J gene segment comprises said JH6*02 gene segment.
 14. The cell of claim 3, wherein said J gene segments comprise a plurality of J gene segments comprising a 3′ J gene segment positioned downstream in said plurality and upstream of said constant region in said locus, and said 3′ J gene segment comprises said JH6*02 gene segment.
 15. The vertebrate of claim 1, wherein said locus comprises one, more or all human D gene segments D3-9; D4-17; D3-10; D2-2; D5-24; D6-19; D3-22; D6-13; D5-12; D1-26; D1-20; D5-18; D3-16; D2-21; D1-14; D7-27; D1-1; D6-25; D2-14; and D4-23.
 16. The cell of claim 3, wherein the locus comprises one, more or all human D gene segments D3-9; D4-17; D3-10; D2-2; D5-24; D6-19; D3-22; D6-13; D5-12; D1-26; D1-20; D5-18; D3-16; D2-21; D1-14; D7-27; D1-1; D6-25; D2-14; and D4-23.
 17. The vertebrate of claim 15, wherein the locus comprises one, more or all human D gene segments D3-9, D3-10, D6-19, D4-17, D6-13, D3-22, D2-2, D2-25 and D3-3.
 18. The cell of claim 16, wherein the locus comprises one, more or all human D gene segments D3-9, D3-10, D6-19, D4-17, D6-13, D3-22, D2-2, D2-25 and D3-3.
 19. The vertebrate of claim 1, wherein the locus comprises a plurality of human D gene segments and said JH6*02 is in human germline configuration with respect to the 3′-most human D gene segment.
 20. The cell of claim 3, wherein the locus comprises a plurality of human D gene segments and the JH6*02 is in human germline configuration with respect to the 3′-most human D gene segment.
 21. The vertebrate of claim 1, wherein the locus comprises one, more or all of IGHV gene segments selected from V3-21, V3-13, V3-7, V6-1, V1-8, V1-2, V7-4-1, V1-3, V1-18, V4-4, V3-9, V3-23, V3-11 and V3-20.
 22. The cell of claim 3, wherein the locus comprises one, more or all of IGHV gene segments selected from V3-21, V3-13, V3-7, V6-1, V1-8, V1-2, V7-4-1, V1-3, V1-18, V4-4, V3-9, V3-23, V3-11 and V3-20.
 23. The vertebrate of claim 1, wherein the locus comprises one, more or all of human D3-9*01, D3-10*01, D6-19*01, D6-13*01, D1-26*01, IGHV1-8*01, IGHV4-61*01, IGHV6-1*01, IGHV4-4*02, IGHV1-3*01, IGHV3-66*03, IGHV3-7*01 and IGHV3-9*01.
 24. The cell of claim 3, wherein the locus comprises one, more or all of human D3-9*01, D3-10*01, D6-19*01, D6-13*01, D1-26*01, IGHV1-8*01, IGHV4-61*01, IGHV6-1*01, IGHV4-4*02, IGHV1-3*01, IGHV3-66*03, IGHV3-7*01 and IGHV3-9*01.
 25. An antibody-producing cell that is a progeny of the cell of claim 3, wherein the antibody-producing cell comprises a heavy chain locus comprising a rearranged variable region produced by recombination of human JH6*02 with a D gene segment and a VH gene segment.
 26. The cell of claim 25, which is a B-cell or hybridoma that expresses a target antigen-specific antibody comprising a heavy chain that comprises a rearranged variable region encoded by a recombined Vh-D-human JH6*02 gene produced by recombination of said gene segments with said human JH6*02.
 27. The vertebrate of claim 1, wherein the antibody heavy chain specifically binds a target antigen.
 28. The cell of claim 3, wherein the antibody heavy chain specifically binds a target antigen.
 29. The vertebrate of claim 1, wherein said antibody heavy chain comprises a variable region comprising a HCDR3 length of at least 20 amino acids.
 30. The cell of claim 3, wherein said antibody heavy chain comprises a variable region comprising a HCDR3 length of at least 20 amino acids.
 31. The vertebrate of claim 1, wherein said antibody heavy chain is a product of the recombination of JH6*02 with a human VH gene segment recited in claim 21 or 23 and/or a D gene segment recited in claim 15, 17 or
 23. 32. The cell of claim 3, wherein said antibody heavy chain is a product of the recombination of JH6*02 with a human VH gene segment recited in claim 22 or 24 and/or a D gene segment recited in claim 16, 18 or
 24. 33. The vertebrate of claim 1, wherein said genome comprises endogenous heavy chain variable region gene segments which are nonfunctional to produce endogenous Ig heavy chain.
 34. The cell of claim 3, wherein said genome comprises endogenous heavy chain variable region gene segments which are nonfunctional to produce endogenous Ig heavy chain.
 35. The vertebrate of claim 1, wherein the genome is homozygous for said heavy chain locus.
 36. The cell of claim 3, wherein the genome is homozygous for said heavy chain locus.
 37. The vertebrate of claim 1, wherein the constant region is an endogenous constant region.
 38. The cell of claim 3, wherein the constant region is an endogenous constant region.
 39. A method for producing an antibody specific to a target antigen, the method comprising immunizing the non-human vertebrate according to claim 1 with said antigen, thereby producing said antibody specific to said target antigen, wherein said antibody comprises a heavy chain variable region comprising an HCDR3, and wherein said heavy chain variable region is a product of the recombination of human JH6*02 with a VH and a D gene segment.
 40. The method of claim 39, further comprising the step of isolating said antibody.
 41. The method of claim 40, wherein the constant region is an endogenous constant region.
 42. A method for producing a human an antibody comprising carrying out the method of claim 40 or 41, wherein the constant region of the locus is a non-human vertebrate constant region, further comprising the step of replacing the non-human constant region of the isolated antibody with a human constant region.
 43. The method of claim 39, further comprising the step of isolating a B cell from said mouse, wherein said B cell expresses said antibody.
 44. The method of claim 43, further comprising the step of producing a hybridoma from said B cell.
 45. The method of claim 44, further comprising the step of isolating said antibody from said hybridoma.
 46. The method of claim 39, wherein the non-constant region is an endogenous constant region.
 47. The method of claim 46, further comprising the step of isolating a B cell from said mouse, wherein said B cell expresses said antibody.
 48. The method of claim 47, further comprising the step of producing a hybridoma from said B cell, wherein said hybridoma expresses said antibody.
 49. The method of claim 48, further comprising the step of isolating said antibody from said hybridoma.
 50. A method for producing a heavy chain or VH domain thereof, of an antibody specific to a target antigen, the method comprising immunizing a non-human vertebrate according to claim 1 with the antigen, thereby producing said heavy chain or VH domain thereof, of an antibody specific to said target antigen, wherein the heavy chain or VH domain thereof comprises an HCDR3, and wherein said VH domain is a product of the recombination of human JH6*02 with a VH and a D gene segment.
 51. The method of claim 50, further comprising the step of isolating said heavy chain or VH domain thereof.
 52. The method of claim 51, wherein the constant region is an endogenous constant region.
 53. The method of claim 50, 51 or 52, wherein the heavy chain or VH domain thereof, comprises mouse AID-pattern somatic hypermutations and/or mouse dTd-pattern mutations.
 54. The method of claim 39 or 46, wherein the heavy chain or VH domain thereof, of said antibody comprises mouse AID-pattern somatic hypermutations and/or mouse dTd-pattern mutations.
 55. A method for producing a human an antibody comprising carrying out the method of claim 40 or 41, wherein the constant region of the locus is a non-human vertebrate constant region, wherein the heavy chain or VH domain of said antibody comprises mouse AID-pattern somatic hypermutations and/or mouse dTd-pattern mutations, further comprising the step of replacing the non-human constant region of the isolated antibody with a human constant region.
 56. The method of claim 50, further comprising the step of operably linking the VH domain with a human constant domain. 